Isolated human phospholipase proteins, nucleic acid molecules encoding human phospholipase proteins, and uses thereof

ABSTRACT

The present invention provides amino acid sequences of peptides that are encoded by genes within the human genome, the phospholipase peptides of the present invention. The present invention specifically provides isolated peptide and nucleic acid molecules, methods of identifying orthologs and paralogs of the phospholipase peptides, and methods of identifying modulators of the phospholipase peptides.

RELATED APPLICATIONS

The present application is a divisional of U.S. application Ser. No.09/801,052, filed on Mar. 8, 2001 and issued on Apr. 9, 2002 as U.S.Pat. No. 6,368,842, which claims priority to U.S. ProvisionalApplication No. 60/255, 386, filed on Dec. 15, 2000.

FIELD OF THE INVENTION

The present invention is in the field of phospholipase proteins that arerelated to the phospholipase D subfamily, recombinant DNA molecules, andprotein production. The present invention specifically provides novelpeptides and proteins that effect protein phosphorylation and nucleicacid molecules encoding such peptide and protein molecules, all of whichare useful in the development of human therapeutics and diagnosticcompositions and methods.

BACKGROUND OF THE INVENTION Phospholiipases

There are three major families of known human phospholipase enzymes:Phospholipase A2, Phospholipase C, and Phospholipase D.

Enzymes in the Phospholipase A2 family (“PlA2”) hydrolyze the sn-2 fattyacid acyl ester bond of phosphoglycerides, releasing free fatty acidsand lysophospholipids. The PlA2s constitute a diverse family of enzymeswith respect to sequence, function, localization and divalent cationrequirements. They play an important role in a variety of cellularprocesses, including the digestion and metabolism of phospholipids aswell as the production of precursors for inflammatory reactions. ThePlA2s have been classified into at least 5 groups (although differentclassification schemes exist and up to 10 groups have been identified bysome authorities) based on their size, structure and need for divalentcations. Groups I, II and III all contain secreted forms of PlA, whichare extracellular enzymes that have a low molecular mass and requirecalcium ions for catalysis. Groups IV and V contain cytosolic forms ofPlA2s that have a high molecular mass and do not necessarily requirecalcium ions.

Amongst the best characterized of the PlA2 phospholipases are digestiveenzymes secreted as zymogens by the pancreas. These enzymes, which areinvolved in the hydrolysis of dietary phospholipids, have stronghomology to the venom phospholipases of snakes. Other PlA2s playimportant roles in the control of signaling cascades such as thecytosolic PlA2, Group IVA enzyme (“PLA2G4A”) which catalyzes the releaseof arachidonic acid from membrane phospholipids. Arachidonic acid servesas a precursor for a wide spectrum of biological effectors, collectivelyknown as eicosanoids (and including the prostaglandin group ofmolecules) that are involved in hemodynamic regulation, inflammatoryresponses and other cellular processes.

Another biologically active phospholipid, platelet-activating factor(“PAF”) is hydrolyzed to metabolically-inactive degradation products bythe group VII PlA2 known as PAF acetylhydrolase. Deficiency of PAFacetylhydrolase has been reported in patients with systemic lupuserythematosis and increased levels of PAF have been reported in childrenwith acute asthmatic attacks. Elevated levels of the group II PlA2 knownas PLA2G2A have been reported in plasma and synovial fluid in patientswith inflammatory arthritis. Studies of a mouse colon cancer modelshowed that alleles of the murine ortholog of this gene were able tomodify the number of tumors that developed in animals with multipleintestinal neoplasia (a mouse model of the human disorder known asfamilial adenomtous polyposis). Subsequent studies in humans showedmutations in PLA2G2A were associated with the risk of developingcolorectal cancer. PLA2G2A is presumed to act through altering cellularmicroenvironments within the intestinal crypts of the colonic mucosa,although the precise mechanism by which this effect is exerted is notclear.

Enzymes in the Phospholipase C (“PLC”) family catalyze the hydrolysis ofthe plasma membrane phospholipids, phosphatidyl inositol phosphate(“PIP”) or phosphatidylinositol 4,5-biphosphate (“PIP2”), generating asproducts the second messengers, 1,4,5-inositol triphosphate (“IP3”) and1,2-diacylglycerol (“DAG”). Molecules belonging to the PLC gene familyare divided into subfamilies, PLC-beta, PLC-gamma and PLC-delta.PLC-delta is distinguished from PLC-gamma by lack of the SH2 and SH3domains that are essential for activation of PLC-gamma by tyrosineprotein kinases. PLC-delta is distinguished from PLC-beta by lack of theC-terminal region of PLC-beta that is responsible for binding andactivation of G proteins. Various PLC enzymes play important roles insignal transduction cascades throughout the body. Activating signalsinclude hormones, growth factors and neurotransmitters. One of thefunctions of IP2 is to modulate intracellular calcium levels while-DAGis involved in the activation of certain protein kinases and can promotemembrane fusion in processes involving vesicular trafficking.

Enzymes in the Phospholipase D (“PLD”) family catalyze the hydrolysis ofphosphatidylcholine (“PC”) and other phospholipids to producephosphatidic acid. A range of agonists acting through G protein-coupledreceptors and receptor tyrosine kinases stimulate this hydrolysis.Phosphatidic acid appears to be important as a second messenger capableof activating a diverse range of signaling pathways. PC-specific PLDactivity has been implicated in numerous cellular pathways, includingsignal transduction, membrane trafficking, the regulation of mitosis,regulated secretion, cytoskeletal reorganization, transcriptionalregulation and cell-cycle control. Many proteins ate attached to theplasma membrane via a glysylphosphatidylinositol (“GPI”) anchor.Phosphatidylinositol-glycan (“PIG”)-specific PLDs selectively hydrolyzethe inositol phosphate linkage, allowing release of the protein.

Phospholipase D

The protein provided by the present invention is a novel humanphospholipase splice form that is related to the phospholipase D (PLD)family. In particular, the novel phospholipase splice form provided bythe present invention lacks exon 2 found in a prior art phospholipaseprotein (patent seq W57899). PLD proteins are known to exist asalternative splice forms. For example, alternate splice variants of twoPLD isoforms, termed PLD1 and PLD2, have previously been identified(Steed et al., FASEB J. 1998 Oct;12(13):1309-17).

The phospholipase D family is characterized by a conserved HXKXXXXDmotif and this characteristic motif is essential for the catalyticfunction of PLD. A subclass of PLD exists that is characterized by asecond HXKXXXXD motif with a conserved Asp to Glu substitution.

PLD enzymes play important roles in signal transduction and membranevesicular trafficking in mammalian cells (Pedersen et al., J Biol Chem1998 Nov 20;273(47):31494-504). In particular, PLD cleavesphosphatidylcholine in response to cell stimuli, thereby releasingphosphatidic acid, which is involved in numerous cellular responses thatmay play a role in, for example, regulation of secretion, mitogenesis,or cytoskeletal changes (Steed et al., FASEB J. 1998Oct;12(13):1309-17).

The activity and regulation of recombinant human PLD2 are identical tothat of recombinant mouse PLD2. Analysis of the amino acid sequences ofthe human PLD1 and PLD2 isoforms revealed Pleckstrin homology domains.(Steed et al., FASEB J. 1998 Oct;12(13): 1309-17). Orthologs of PLD mayexist in vaccinia virus (Pedersen et al., J Biol Chem 1998 Nov20;273(47):31494-504).

A murine PLD gene, termed sam-9 gene, has been found to be expressed athigh levels in the brain, particularly in mature neurons of theforebrain, and the gene is turned on during late stages of neurogenesis(Pedersen et al., J Biol Chem 1998 Nov 20;273(47):31494-504).

Phospholipase proteins, particularly members of the phospholipase Dsubfamily, are a major target for drug action and development.Accordingly, it is valuable to the field of pharmaceutical developmentto identify and characterize previously unknown members of thissubfamily of phospholipase proteins. The present invention advances thestate of the art by providing previously unidentified humanphospholipase proteins that have homology to members of thephospholipase D subfamily.

SUMMARY OF THE INVENTION

The present invention is based in part on the identification of aminoacid sequences of human phospholipase peptides and proteins that arerelated to the phospholipase D subfamily, as well as allelic variantsand other mammalian orthologs thereof. These unique peptide sequences,and nucleic acid sequences that encode these peptides, can be used asmodels for the development of human therapeutic targets, aid in theidentification of therapeutic proteins, and serve as targets for thedevelopment of human therapeutic agents that modulate phospholipaseactivity in cells and tissues that express the phospholipase.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes.

DESCRIPTION OF THE FIGURE SHEETS

FIG. 1 provides the nucleotide sequence of a cDNA molecule or transcriptsequence that encodes the phospholipase protein of the presentinvention. (SEQ ID NO:1) In addition, structure and functionalinformation is provided, such as ATG start, stop and tissuedistribution, where available, that allows one to readily determinespecific uses of inventions based on this molecular sequence.Experimental data as provided in FIG. I indicates expression in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes.

FIG. 2 provides the predicted amino acid sequence of the phospholipaseof the present invention. (SEQ ID NO:2) In addition structure andfunctional information such as protein family, function, andmodification sites is provided where available, allowing one to readilydetermine specific uses of inventions based on this molecular sequence.

FIG. 3 provides genomic sequences that span the gene encoding thephospholipase protein of the present invention. (SEQ ID NO:3) Inaddition structure and functional information, such as intron/exonstructure, promoter location, etc., is provided where available,allowing one to readily determine specific uses of inventions based onthis molecular sequence. As illustrated in FIG. 3, SNPs were identifiedat nine different nucleotide positions.

DETAILED DESCRIPTION OF THE INVENTION General Description

The present invention is based on the sequencing of the human genome.During the sequencing and assembly of the human genome, analysis of thesequence information revealed previously unidentified fragments of thehuman genome that encode peptides that share structural and/or sequencehomology to protein/peptide/domains identified and characterized withinthe art as being a phospholipase protein or part of a phospholipaseprotein and are related to the phospholipase D subfamily. Utilizingthese sequences, additional genomic sequences were assembled andtranscript and/or cDNA sequences were isolated and characterized. Basedon this analysis, the present invention provides amino acid sequences ofhuman phospholipase peptides and proteins that are related to thephospholipase D subfamily, nucleic acid sequences in the form oftranscript sequences, cDNA sequences and/or genomic sequences thatencode these phospholipase peptides and proteins, nucleic acid variation(allelic information), tissue distribution of expression, andinformation about the closest art known protein/peptide/domain that hasstructural or sequence homology to the phospholipase of the presentinvention.

In addition to being previously unknown, the peptides that are providedin the present invention are selected based on their ability to be usedfor the development of commercially important products and services.Specifically, the present peptides are selected based on homology and/orstructural relatedness to known phospholipase proteins of thephospholipase D subfamily and the expression pattern observed.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. The art has clearly establishedthe commercial importance of members of this family of proteins andproteins that have expression patterns similar to that of the presentgene. Some of the more specific features of the peptides of the presentinvention, and the uses thereof, are described herein, particularly inthe Background of the Invention and in the annotation provided in theFigures, and/or are known within the art for each of the knownphospholipase D family or subfamily of phospholipase proteins.

Specific Embodiments

Peptide Molecules

The present invention provides nucleic acid sequences that encodeprotein molecules that have been identified as being members of thephospholipase family of proteins and are related to the phospholipase Dsubfamily (protein sequences are provided in FIG. 2, transcript/cDNAsequences are provided in FIG. 1 and genomic sequences are provided inFIG. 3). The peptide sequences provided in FIG. 2, as well as theobvious variants described herein, particularly allelic variants asidentified herein and using the information in FIG. 3, will be referredherein as the phospholipase peptides of the present invention,phospholipase peptides, or peptides/proteins of the present invention.

The present invention provides isolated peptide and protein moleculesthat consist of, consist essentially of, or comprise the amino acidsequences of the phospholipase peptides disclosed in the FIG. 2,(encoded by the nucleic acid molecule shown in FIG. 1, transcript/cDNAor FIG. 3, genomic sequence), as well as all obvious variants of thesepeptides that are within the art to make and use. Some of these variantsare described in detail below.

As used herein, a peptide is said to be “isolated” or “purified” when itis substantially free of cellular material or free of chemicalprecursors or other chemicals. The peptides of the present invention canbe purified to homogeneity or other degrees of purity. The level ofpurification will be based on the intended use. The critical feature isthat the preparation allows for the desired function of the peptide,even if in the presence of considerable amounts of other components (thefeatures of an isolated nucleic acid molecule is discussed below).

In some uses, “substantially free of cellular material” includespreparations of the peptide having less than about 30% (by dry weight)other proteins (i.e., contaminating protein), less than about 20% otherproteins, less than about 10% other proteins, or less than about 5%other proteins. When the peptide is recombinantly produced, it can alsobe substantially free of culture medium, i.e., culture medium representsless than about 20% of the volume of the protein preparation.

The language “substantially free of chemical precursors or otherchemicals” includes preparations of the peptide in which it is separatedfrom chemical precursors or other chemicals that are involved in itssynthesis. In one embodiment, the language “substantially free ofchemical precursors or other chemicals” includes preparations of thephospholipase peptide having less than about 30% (by dry weight)chemical precursors or other chemicals, less than about 20% chemicalprecursors or other chemicals, less than about 10% chemical precursorsor other chemicals, or less than about 5% chemical precursors or otherchemicals.

The isolated phospholipase peptide can be purified from cells thatnaturally express it, purified from cells that have been altered toexpress it (recombinant), or synthesized using known protein synthesismethods. Experimental data as provided in FIG. 1 indicates expression inhumans in the brain (including neuroblastomas and infant brain), uterus,lung, ovary adenocarcinomas, and leukocytes. For example, a nucleic acidmolecule encoding the phospholipase peptide is cloned into an expressionvector, the expression vector introduced into a host cell and theprotein expressed in the host cell. The protein can then be isolatedfrom the cells by an appropriate purification scheme using standardprotein purification techniques. Many of these techniques are describedin detail below.

Accordingly, the present invention provides proteins that consist of theamino acid sequences provided in FIG. 2 (SEQ ID NO:2), for example,proteins encoded by the transcript/cDNA nucleic acid sequences shown inFIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQID NO:3). The amino acid sequence of such a protein is provided in FIG.2. A protein consists of an amino acid sequence when the amino acidsequence is the final amino acid sequence of the protein.

The present invention further provides proteins that consist essentiallyof the amino acid sequences provided in FIG. 2 (SEQ ID NO:2), forexample, proteins encoded by the transcript/cDNA nucleic acid sequencesshown in FIG. 1 (SEQ ID NO:1) and the genomic sequences provided in FIG.3 (SEQ ID NO:3). A protein consists essentially of an amino acidsequence when such an amino acid sequence is present with only a fewadditional amino acid residues, for example from about 1 to about 100 orso additional residues, typically from 1 to about 20 additional residuesin the final protein.

The present invention further provides proteins that comprise the aminoacid sequences provided in FIG. 2 (SEQ ID NO:2), for example, proteinsencoded by the transcript/cDNA nucleic acid sequences shown in FIG. 1(SEQ ID NO:1) and the genomic sequences provided in FIG. 3 (SEQ IDNO:3). A protein comprises an amino acid sequence when the amino acidsequence is at least part of the final amino acid sequence of theprotein. In such a fashion, the protein can be only the peptide or haveadditional amino acid molecules, such as amino acid residues (contiguousencoded sequence) that are naturally associated with it or heterologousamino acid residues/peptide sequences. Such a protein can have a fewadditional amino acid residues or can comprise several hundred or moreadditional amino acids. The preferred classes of proteins that arecomprised of the phospholipase peptides of the present invention are thenaturally occurring mature proteins. A brief description of how varioustypes of these proteins can be made/isolated is provided below.

The phospholipase peptides of the present invention can be attached toheterologous sequences to form chimeric or fusion proteins. Suchchimeric and fusion proteins comprise a phospholipase peptideoperatively linked to a heterologous protein having an amino acidsequence not substantially homologous to the phospholipase peptide.“Operatively linked” indicates that the phospholipase peptide and theheterologous protein are fused in-frame. The heterologous protein can befused to the N-terminus or C-terminus of the phospholipase peptide.

In some uses, the fusion protein does not affect the activity of thephospholipase peptide per se. For example, the fusion protein caninclude, but is not limited to, enzymatic fusion proteins, for examplebeta-galactosidase fusions, yeast two-hybrid GAL fusions, poly-Hisfusions, MYC-tagged, HI-tagged and Ig fusions. Such fusion proteins;particularly poly-His fusions, can facilitate the purification ofrecombinant phospholipase peptide. In certain host cells (e.g.,mammalian host cells), expression and/or secretion of a protein can beincreased by using a heterologous signal sequence.

A chimeric or fusion protein can be produced by standard recombinant DNAtechniques. For example, DNA fragments coding for the different proteinsequences are ligated together in-frame in accordance with conventionaltechniques. In another embodiment, the fusion gene can be synthesized byconventional techniques including automated DNA synthesizers.Alternatively, PCR amplification of gene fragments can be carried outusing anchor primers which give rise to complementary overhangs betweentwo consecutive gene fragments which can subsequently be annealed andre-amplified to generate a chimeric gene sequence (see Ausubel et al.,Current Protocols in Molecular Biology, 1992). Moreover, many expressionvectors are commercially available that already encode a fusion moiety(e.g., a GST protein). A phospholipase peptide-encoding nucleic acid canbe cloned into such an expression vector such that the fusion moiety islinked in-frame to the phospholipase peptide.

As mentioned above, the present invention also provides and enablesobvious variants of the amino acid sequence of the proteins of thepresent invention, such as naturally occurring mature forms of thepeptide, allelic/sequence variants of the peptides, non-naturallyoccurring recombinantly derived variants of the peptides, and orthologsand paralogs of the peptides. Such variants can readily be generatedusing art-known techniques in the fields of recombinant nucleic acidtechnology and protein biochemistry. It is understood, however, thatvariants exclude any amino acid sequences disclosed prior to theinvention.

Such variants can readily be identified/made using molecular techniquesand the sequence information disclosed herein. Further, such variantscan readily be distinguished from other peptides based on sequenceand/or structural homology to the phospholipase peptides of the presentinvention. The degree of homology/identity present will be basedprimarily on whether the peptide is a functional variant ornon-functional variant, the amount of divergence present in the paralogfamily and the evolutionary distance between the orthologs.

To determine the percent identity of two amino acid sequences or twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes (e.g., gaps can be introduced in one or both of a first and asecond amino acid or nucleic acid sequence for optimal alignment andnon-homologous sequences can be disregarded for comparison purposes). Ina preferred embodiment, at least 30%, 40%, 50%, 60%, 70%, 80%, or 90% ormore of the length of a reference sequence is aligned for comparisonpurposes. The amino acid residues or nucleotides at corresponding aminoacid positions or nucleotide positions are then compared. When aposition in the first sequence is occupied by the same amino acidresidue or nucleotide as the corresponding position in the secondsequence, then the molecules are identical at that position (as usedherein amino acid or nucleic acid “identity” is equivalent to amino acidor nucleic acid “homology”). The percent identity between the twosequences is a function of the number of identical positions shared bythe sequences, taking into account the number of gaps, and the length ofeach gap, which need to be introduced for optimal alignment of the twosequences.

The comparison of sequences and determination of percent identity andsimilarity between two sequences can be accomplished using amathematical algorithm. (Computational Molecular Biology, Lesk, A. M.,ed., Oxford University Press, New York, 1988; Biocomputing: Informaticsand Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993;Computer Analysis of Sequence Data, Part 1, Griffin, A. M., and Griffin,H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis inMolecular Biology, von Heinje, G., Academic Press, 1987; and SequenceAnalysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press,New York, 1991). In a preferred embodiment, the percent identity betweentwo amino acid sequences is determined using the Needleman and Wunsch(J. Mol. Biol. (48):444-453 (1970)) algorithm which has beenincorporated into the GAP program in the GCG software package (availableat http://www.gcg.com), using either a Blossom 62 matrix or a PAM250matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a lengthweight of 1, 2, 3, 4, 5, or 6. In yet another preferred embodiment, thepercent identity between two nucleotide sequences is determined usingthe GAP program in the GCG software package (Devereux, J., et al.,Nucleic Acids Res. 12(1):387 (1984)) (available at http://www.gcg.com),using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80and a length weight of 1, 2, 3, 4, 5, or 6. In another embodiment, thepercent identity between two amino acid or nucleotide sequences isdetermined using the algorithm of E. Myers and W. Miller (CABIOS,4:11-17 (1989)) which has been incorporated into the ALIGN program(version 2.0), using a PAM120 weight residue table, a gap length penaltyof 12 and a gap penalty of 4.

The nucleic acid and protein sequences of the present invention canfurther be used as a “query sequence” to perform a search againstsequence databases to, for example, identify other family members orrelated sequences. Such searches can be performed using the NBLAST andXBLAST programs (version 2.0) of Altschul, et al. (J. Mol. Biol.215:403-10 (1990)). BLAST nucleotide searches can be performed with theNBLAST program, score=100, wordlength=12 to obtain nucleotide sequenceshomologous to the nucleic acid molecules of the invention. BLAST proteinsearches can be performed with the XBLAST program, score=50,wordlength=3 to obtain amino acid sequences homologous to the proteinsof the invention. To obtain gapped alignments for comparison purposes,Gapped BLAST can be utilized as described in Altschul et al. (NucleicAcids Res. 25(17):3389-3402 (1997)). When utilizing BLAST and gappedBLAST programs, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used.

Full-length pre-processed forms, as well as mature processed forms, ofproteins that comprise one of the peptides of the present invention canreadily be identified as having complete sequence identity to one of thephospholipase peptides of the present invention as well as being encodedby the same genetic locus as the phospholipase peptide provided herein.The gene encoding the novel phospholipase protein of the presentinvention is located on a genome component that has been mapped to humanchromosome 2 (as indicated in FIG. 3), which is supported by multiplelines of evidence, such as STS and BAC map data.

Allelic variants of a phospholipase peptide can readily be identified asbeing a human protein having a high degree (significant) of sequencehomology/identity to at least a portion of the phospholipase peptide aswell as being encoded by the same genetic locus as the phospholipasepeptide provided herein. Genetic locus can readily be determined basedon the genomic information provided in FIG. 3, such as the genomicsequence mapped to the reference human. The gene encoding the novelphospholipase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 2 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data. As used herein, two proteins (or a region of theproteins) have significant homology when the amino acid sequences aretypically at least about 70-80%, 80-90%, and more typically at leastabout 90-95% or more homologous. A significantly homologous amino acidsequence, according to the present invention, will be encoded by anucleic acid sequence that will hybridize to a phospholipase peptideencoding nucleic acid molecule under stringent conditions as more fullydescribed below.

FIG. 3 provides information on SNPs that have been found in the geneencoding the phospholipase proteins of the present invention. SNPs wereidentified at nine different nucleotide positions. SNPs outside the ORFand in introns may affect control/regulatory elements.

Paralogs of a phospholipase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the phospholipase peptide, as being encoded by a gene fromhumans, and as having similar activity or function. Two proteins willtypically be considered paralogs when the amino acid sequences aretypically at least about 60% or greater, and more typically at leastabout 70% or greater homology through a given region or domain. Suchparalogs will be encoded by a nucleic acid sequence that will hybridizeto a phospholipase peptide encoding nucleic acid molecule under moderateto stringent conditions as more fully described below.

Orthologs of a phospholipase peptide can readily be identified as havingsome degree of significant sequence homology/identity to at least aportion of the phospholipase peptide as well as being encoded by a genefrom another organism. Preferred orthologs will be isolated frommammals, preferably primates, for the development of human therapeutictargets and agents. Such orthologs will be encoded by a nucleic acidsequence that will hybridize to a phospholipase peptide encoding nucleicacid molecule under moderate to stringent conditions, as more fullydescribed below, depending on the degree of relatedness of the twoorganisms yielding the proteins.

Non-naturally occurring variants of the phospholipase peptides of thepresent invention can readily be generated using recombinant techniques.Such variants include, but are not limited to deletions, additions andsubstitutions in the amino acid sequence of the phospholipase peptide.For example, one class of substitutions are conserved amino acidsubstitution. Such substitutions are those that substitute a given aminoacid in a phospholipase peptide by another amino acid of likecharacteristics. Typically seen as conservative substitutions are thereplacements, one for another, among the aliphatic amino acids Ala, Val,Leu, and Ile; interchange of the hydroxyl residues Ser and Thr; exchangeof the acidic residues Asp and Glu; substitution between the amideresidues Asn and Gln; exchange of the basic residues Lys and Arg; andreplacements among the aromatic residues Phe and Tyr. Guidanceconcerning which amino acid changes are likely to be phenotypicallysilent are found in Bowie et al., Science 247:1306-1310 (1990).

Variant phospholipase peptides can be fully functional or can lackfunction in one or more activities, e.g. ability to bind substrate,ability to phosphorylate substrate, ability to mediate signaling, etc.Fully functional variants typically contain only conservative variationor variation in non-critical residues or in non-critical regions. FIG. 2provides the result of protein analysis and can be used to identifycritical domains/regions. Functional variants can also containsubstitution of similar amino acids that result in no change or aninsignificant change in function. Alternatively, such substitutions maypositively or negatively affect function to some degree.

Non-functional variants typically contain one or more non-conservativeamino acid substitutions, deletions, insertions, inversions, ortruncation or a substitution, insertion, inversion, or deletion in acritical residue or critical region.

Amino acids that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham et al., Science 244:1081-1085 (1989)),particularly using the results provided in FIG. 2. The latter procedureintroduces single alanine mutations at every residue in the molecule.The resulting mutant molecules are then tested for biological activitysuch as phospholipase activity or in assays such as an in vitroproliferative activity. Sites that are critical for bindingpartner/substrate binding can also be determined by structural analysissuch as crystallization, nuclear magnetic resonance or photoaffinitylabeling (Smith et al., J. Mol. Biol. 224:899-904 (1992); de Vos et al.Science 255:306-312 (1992)).

The present invention further provides fragments of the phospholipasepeptides, in addition to proteins and peptides that comprise and consistof such fragments, particularly those comprising the residues identifiedin FIG. 2. The fragments to which the invention pertains, however, arenot to be construed as encompassing fragments that may be disclosedpublicly prior to the present invention.

As used herein, a fragment comprises at least 8, 10, 12, 14, 16, or morecontiguous amino acid residues from a phospholipase peptide. Suchfragments can be chosen based on the ability to retain one or more ofthe biological activities of the phospholipase peptide or could bechosen for the ability to perform a function, e.g. bind a substrate oract as an immunogen. Particularly important fragments are biologicallyactive fragments, peptides that are, for example, about 8 or more aminoacids in length. Such fragments will typically comprise a domain ormotif of the phospholipase peptide, e.g., active site, a transmembranedomain or a substrate-binding domain. Further, possible fragmentsinclude, but are not limited to, domain or motif containing fragments,soluble peptide fragments, and fragments containing immunogenicstructures. Predicted domains and functional sites are readilyidentifiable by computer programs well known and readily available tothose of skill in the art (e.g., PROSITE analysis). The results of onesuch analysis are provided in FIG. 2.

Polypeptides often contain amino acids other than the 20 amino acidscommonly referred to as the 20 naturally occurring amino acids. Further,many amino acids, including the terminal amino acids, may be modified bynatural processes, such as processing and other post-translationalmodifications, or by chemical modification techniques well known in theart. Common modifications that occur naturally in phospholipase peptidesare described in basic texts, detailed monographs, and the researchliterature, and they are well known to those of skill in the art (someof these features are identified in FIG. 2).

Known modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, covalent attachment of flavin,covalent attachment of a heme moiety, covalent attachment of anucleotide or nucleotide derivative, covalent attachment of a lipid orlipid derivative, covalent attachment of phosphotidylinositol,cross-linking, cyclization, disulfide bond formation, demethylation,formation of covalent crosslinks, formation of cystine, formation ofpyroglutamate, formylation, gamma carboxylation, glycosylation, GPIanchor formation, hydroxylation, iodination, methylation,myristoylation, oxidation, proteolytic processing, phosphorylation,prenylation, racemization, selenoylation, sulfation, transfer-RNAmediated addition of amino acids to proteins such as arginylation, andubiquitination.

Such modifications are well known to those of skill in the art and havebeen described in great detail in the scientific literature. Severalparticularly common modifications, glycosylation, lipid attachment,sulfation, gamma-carboxylation of glutamic acid residues, hydroxylationand ADP-ribosylation, for instance, are described in most basic texts,such as Proteins—Structure and Molecular Properties, 2nd Ed., T. E.Creighton, W. H. Freeman and Company, New York (1993). Many detailedreviews are available on this subject, such as by Wold, F.,Posttranslational Covalent Modification of Proteins, B. C. Johnson, Ed.,Academic Press, New York 1-12 (1983); Seifter et al. (Meth. Enzymol.182: 626-646 (1990)) and Rattan et al. (Ann. N.Y. Acad. Sci. 663:48-62(1992)).

Accordingly, the phospholipase peptides of the present invention alsoencompass derivatives or analogs in which a substituted amino acidresidue is not one encoded by the genetic code, in which a substituentgroup is included, in which the mature phospholipase peptide is fusedwith another compound, such as a compound to increase the half-life ofthe phospholipase peptide, (for example, polyethylene glycol), or inwhich the additional amino acids are fused to the mature phospholipasepeptide, such as a leader or secretory sequence or a sequence forpurification of the mature phospholipase peptide or a pro-proteinsequence.

Protein/Peptide Uses

The proteins of the present invention can be used in substantial andspecific assays related to the functional information provided in theFigures; to raise antibodies or to elicit another immune response; as areagent (including the labeled reagent) in assays designed toquantitatively determine levels of the protein (or its binding partneror ligand) in biological fluids; and as markers for tissues in which thecorresponding protein is preferentially expressed (either constitutivelyor at a particular stage of tissue differentiation or development or ina disease state). Where the protein binds or potentially binds toanother protein or ligand (such as, for example, in aphospholipase-effector protein interaction or phospholipase-ligandinteraction), the protein can be used to identify the bindingpartner/ligand so as to develop a system to identify inhibitors of thebinding interaction. Any or all of these uses are capable of beingdeveloped into reagent grade or kit format for commercialization ascommercial products.

Methods for performing the uses listed above are well known to thoseskilled in the art. References disclosing such methods include“Molecular Cloning: A Laboratory Manual”, 2d ed., Cold Spring HarborLaboratory Press, Sambrook, J., E. F. Fritsch and T. Maniatis eds.,1989, and “Methods in Enzymology: Guide to Molecular CloningTechniques”, Academic Press, Berger, S. L. and A. R. Kimmel eds., 1987.

The potential uses of the peptides of the present invention are basedprimarily on the source of the protein as well as the class/action ofthe protein. For example, phospholipases isolated from humans and theirhuman/mammalian orthologs serve as targets for identifying agents foruse in mammalian therapeutic applications, e.g. a human drug,particularly in modulating a biological or pathological response in acell or tissue that expresses the phospholipase. Experimental data asprovided in FIG. 1 indicates that phospholipase proteins of the presentinvention are expressed in humans in the brain (including neuroblastomasand infant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.Specifically, a virtual northern blot shows expression in the brain(including neuroblastomas and infant brain), uterus, lung, and ovaryadenocarcinomas. In addition, PCR-based tissue screening panels indicateexpression in leukocytes. A large percentage of pharmaceutical agentsare being developed that modulate the activity of phospholipaseproteins, particularly members of the phospholipase D subfamily (seeBackground of the Invention). The structural and functional informationprovided in the Background and Figures provide specific and substantialuses for the molecules of the present invention, particularly incombination with the expression information provided in FIG. 1.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. Such uses can readily bedetermined using the information provided herein, that which is known inthe art, and routine experimentation.

The proteins of the present invention (including variants and fragmentsthat may have been disclosed prior to the present invention) are usefulfor biological assays related to phospholipases that are related tomembers of the phospholipase D subfamily. Such assays involve any of theknown phospholipase functions or activities or properties useful fordiagnosis and treatment of phospholipase-related conditions that arespecific for the subfamily of phospholipases that the one of the presentinvention belongs to, particularly in cells and tissues that express thephospholipase. Experimental data as provided in FIG. 1 indicates thatphospholipase proteins of the present invention are expressed in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. Specifically, a virtual northernblot shows expression in the brain (including neuroblastomas and infantbrain), uterus, lung, and ovary adenocarcinomas. In addition, PCR-basedtissue screening panels indicate expression in leukocytes.

The proteins of the present invention are also useful in drug screeningassays, in cell-based or cell-free systems. Cell-based systems can benative, i.e., cells that normally express the phospholipase, as a biopsyor expanded in cell culture. Experimental data as provided in FIG. 1indicates expression in humans in the brain (including neuroblastomasand infant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.In an alternate embodiment, cell-based assays involve recombinant hostcells expressing the phospholipase protein.

The polypeptides can be used to identify compounds that modulatephospholipase activity of the protein in its natural state or an alteredform that causes a specific disease or pathology associated with thephospholipase. Both the phospholipases of the present invention andappropriate variants and fragments can be used in high-throughputscreens to assay candidate compounds for the ability to bind to thephospholipase. These compounds can be further screened against aftnctional phospholipase to determine the effect of the compound on thephospholipase activity. Further, these compounds can be tested in animalor invertebrate systems to determine activity/effectiveness. Compoundscan be identified that activate (agonist) or inactivate (antagonist) thephospholipase to a desired degree.

Further, the proteins of the present invention can be used to screen acompound for the ability to stimulate or inhibit interaction between thephospholipase protein and a molecule that normally interacts with thephospholipase protein, e.g. a substrate or a component of the signalpathway that the phospholipase protein normally interacts (for example,another phospholipase). Such assays typically include the steps ofcombining the phospholipase protein with a candidate compound underconditions that allow the phospholipase protein, or fragment, tointeract with the target molecule, and to detect the formation of acomplex between the protein and the target or to detect the biochemicalconsequence of the interaction with the phospholipase protein and thetarget, such as any of the associated effects of signal transductionsuch as protein ph6sphorylation, cAMP turnover, and adenylate cyclaseactivation, etc.

Candidate compounds include, for example, 1) peptides such as solublepeptides, including Ig-tailed fusion peptides and members of randompeptide libraries (see, e.g., Lam et al., Nature 354:82-84 (1991);Houghten et al., Nature 354:84-86 (1991)) and combinatorialchemistry-derived molecular libraries made of D- and/or L- configurationamino acids; 2) phosphopeptides (e.g., members of random and partiallydegenerate, directed phosphopeptide libraries, see, e.g., Songyang etal., Cell 72:767-778 (1993)); 3) antibodies (e.g., polyclonal,monoclonal, humanized, anti-idiotypic, chimeric, and single chainantibodies as well as Fab, F(ab′)₂, Fab expression library fragments,and epitope-binding fragments of antibodies); and 4) small organic andinorganic molecules (e.g., molecules obtained from combinatorial andnatural product libraries).

One candidate compound is a soluble fragment of the receptor thatcompetes for substrate binding. Other candidate compounds include mutantphospholipases or appropriate fragments containing mutations that affectphospholipase function and thus compete for substrate. Accordingly, afragment that competes for substrate, for example with a higheraffinity, or a fragment that binds substrate but does not allow release,is encompassed by the invention.

The invention further includes other end point assays to identifycompounds that modulate (stimulate or inhibit) phospholipase activity.The assays typically involve an assay of events in the signaltransduction pathway that indicate phospholipase activity. Thus, thephosphorylation of a substrate, activation of a protein, a change in theexpression of genes that are up- or down-regulated in response to thephospholipase protein dependent signal cascade can be assayed.

Any of the biological or biochemical functions mediated by thephospholipase can be used as an endpoint assay. These include all of thebiochemical or biochemicalibiological events described herein, in thereferences cited herein, incorporated by reference for these endpointassay targets, and other functions known to those of ordinary skill inthe art or that can be readily identified using the information providedin the Figures, particularly FIG. 2. Specifically, a biological functionof a cell or tissues that expresses the phospholipase can be assayed.Experimental data as provided in FIG. 1 indicates that phospholipaseproteins of the present invention are expressed in humans in the brain(including neuroblastomas and infant brain), uterus, lung, ovaryadenocarcinomas, and leukocytes. Specifically, a virtual northern blotshows expression in the brain (including neuroblastomas and infantbrain), uterus, lung, and ovary adenocarcinomas. In addition, PCR-basedtissue screening panels indicate expression in leukocytes.

Binding and/or activating compounds can also be screened by usingchimeric phospholipase proteins in which the amino terminalextracellular domain, or parts thereof, the entire transmembrane domainor subregions, such as any of the seven transmembrane segments or any ofthe intracellular or extracellular loops and the carboxy terminalintracellular domain, or parts thereof, can be replaced by heterologousdomains or subregions. For example, a substrate-binding region can beused that interacts with a different substrate then that which isrecognized by the native phospholipase. Accordingly, a different set ofsignal transduction components is available as an end-point assay foractivation. This allows for assays to be performed in other than thespecific host cell from which the phospholipase is derived.

The proteins of the present invention are also useful in competitionbinding assays in methods designed to discover compounds that interactwith the phospholipase (e.g. binding partners and/or ligands). Thus, acompound is exposed to a phospholipase polypeptide under conditions thatallow the compound to bind or to otherwise interact with thepolypeptide. Soluble phospholipase polypeptide is also added to themixture. If the test compound interacts with the soluble phospholipasepolypeptide, it decreases the amount of complex formed or activity fromthe phospholipase target. This type of assay is particularly useful incases in which compounds are sought that interact with specific regionsof the phospholipase. Thus, the soluble polypeptide that competes withthe target phospholipase region is designed to contain peptide sequencescorresponding to the region of interest.

To perform cell free drug screening assays, it is sometimes desirable toimmobilize either the phospholipase protein, or fragment, or its targetmolecule to facilitate separation of complexes from uncomplexed forms ofone or both of the proteins, as well as to accommodate automation of theassay.

Techniques for immobilizing proteins on matrices can be used in the drugscreening assays. In one embodiment, a fusion protein can be providedwhich adds a domain that allows the protein to be bound to a matrix. Forexample, glutathione-S-transferase fusion proteins can be adsorbed ontoglutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) orglutathione derivatized microtitre plates, which are then combined withthe cell lysates (e.g., ³⁵S-labeled) and the candidate compound, and themixture incubated under conditions conducive to complex formation (e.g.,at physiological conditions for salt and pH). Following incubation, thebeads are washed to remove any unbound label, and the matrix immobilizedand radiolabel determined directly, or in the supernatant after thecomplexes are dissociated. Alternatively, the complexes can bedissociated from the matrix, separated by SDS-PAGE, and the level ofphospholipase-binding protein found in the bead fraction quantitatedfrom the gel using standard electrophoretic techniques. For example,either the polypeptide or its target molecule can be immobilizedutilizing conjugation of biotin and streptavidin using techniques wellknown in the art. Alternatively, antibodies reactive with the proteinbut which do not interfere with binding of the protein to its targetmolecule can be derivatized to the wells of the plate, and the proteintrapped in the wells by antibody conjugation. Preparations of aphospholipase-binding protein and a candidate compound are incubated inthe phospholipase protein-presenting wells and the amount of complextrapped in the well can be quantitated. Methods for detecting suchcomplexes, in addition to those described above for the GST-immobilizedcomplexes, include immunodetection of complexes using antibodiesreactive with the phospholipase protein target molecule, or which arereactive with phospholipase protein and compete with the targetmolecule, as well as enzyme-linked assays which rely on detecting anenzymatic activity associated with the target molecule.

Agents that modulate one of the phospholipases of the present inventioncan be identified using one or more of the above assays, alone or incombination. It is generally preferable to use a cell-based or cell freesystem first and then confirm activity in an animal or other modelsystem. Such model systems are well known in the art and can readily beemployed in this context.

Modulators of phospholipase protein activity identified according tothese drug screening assays can be used to treat a subject with adisorder mediated by the phospholipase pathway, by treating cells ortissues that express the phospholipase. Experimental data as provided inFIG. 1 indicates expression in humans in the brain (includingneuroblastomas and infant brain), uterus, lung, ovary adenocarcinomas,and leukocytes. These methods of treatment include the steps ofadministering a modulator of phospholipase activity in a pharmaceuticalcomposition to a subject in need of such treatment, the modulator beingidentified as described herein.

In yet another aspect of the invention, the phospholipase proteins canbe used as “bait proteins” in a two-hybrid assay or three-hybrid assay(see, e.g., U.S. Pat. No. 5,283,317; Zervos et al. (1993) Cell72:223-232; Madura et al. (1993) J. Biol. Chem. 268:12046-12054; Bartelet al. (1993) Biotechniques 14:920-924; Iwabuchi et al. (1993) Oncogene8:1693-1696; and Brent WO94/10300), to identify other proteins, whichbind to or interact with the phospholipase and are involved inphospholipase activity. Such phospholipase-binding proteins are alsolikely to be involved in the propagation of signals by the phospholipaseproteins or phospholipase targets as, for example, downstream elementsof a phospholipase-mediated signaling pathway. Alternatively, suchphospholipase-binding proteins are likely to be phospholipaseinhibitors.

The two-hybrid system is based on the modular nature of mosttranscription factors, which consist of separable DNA-binding andactivation domains. Briefly, the assay utilizes two different DNAconstructs. In one construct, the gene that codes for a phospholipaseprotein is fused to a gene encoding the DNA binding domain of a knowntranscription factor (e.g., GAL-4). In the other construct, a DNAsequence, from a library of DNA sequences, that encodes an unidentifiedprotein (“prey” or “sample”) is fused to a gene that codes for theactivation domain of the known transcription factor. If the “bait” andthe “prey” proteins are able to interact, in vivo, forming aphospholipase-dependent complex, the DNA-binding and activation domainsof the transcription factor are brought into close proximity. Thisproximity allows transcription of a reporter gene (e.g., LacZ) which isoperably linked to a transcriptional regulatory site responsive to thetranscription factor. Expression of the reporter gene can be detectedand cell colonies containing the functional transcription factor can beisolated and used to obtain the cloned gene which encodes the proteinwhich interacts with the phospholipase protein.

This invention further pertains to novel agents identified by theabove-described screening assays. Accordingly, it is within the scope ofthis invention to further use an agent identified as described herein inan appropriate animal model. For example, an agent identified asdescribed herein (e.g., a phospholipase-modulating agent, an antisensephospholipase nucleic acid molecule, a phospholipase-specific antibody,or a phospholipase-binding partner) can be used in an animal or othermodel to determine the efficacy, toxicity, or side effects of treatmentwith such an agent. Alternatively, an agent identified as describedherein can be used in an animal or other model to determine themechanism of action of such an agent. Furthermore, this inventionpertains to uses of novel agents identified by the above-describedscreening assays for treatments as described herein.

The phospholipase proteins of the present invention are also useful toprovide a target for diagnosing a disease or predisposition to diseasemediated by the peptide. Accordingly, the invention provides methods fordetecting the presence, or levels of, the protein (or encoding mRNA) ina cell, tissue, or organism. Experimental data as provided in FIG. 1indicates expression in humans in the brain (including neuroblastomasand infant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.The method involves contacting a biological sample with a compoundcapable of interacting with the phospholipase protein such that theinteraction can be detected. Such an assay can be provided in a singledetection format or a multi-detection format such as an antibody chiparray.

One agent for detecting a protein in a sample is an antibody capable ofselectively binding to protein. A biological sample includes tissues,cells and biological-fluids isolated from a subject, as well as tissues,cells and fluids present within a subject.

The peptides of the present invention also provide targets fordiagnosing active protein activity, disease, or predisposition todisease, in a patient having a variant peptide, particularly activitiesand conditions that are known for other members of the family ofproteins to which the present one belongs. Thus, the peptide can beisolated from a biological sample and assayed for the presence of agenetic mutation that results in aberrant peptide. This includes aminoacid substitution, deletion, insertion, rearrangement, (as the result ofaberrant splicing events), and inappropriate post-translationalmodification. Analytic methods include altered electrophoretic mobility,altered tryptic peptide digest, altered phospholipase activity incell-based or cell-free assay, alteration in substrate orantibody-binding pattern, altered isoelectric point, direct amino acidsequencing, and any other of the known assay techniques useful fordetecting mutations in a protein. Such an assay can be provided in asingle detection format or a multi-detection format such as an antibodychip array.

In vitro techniques for detection of peptide include enzyme linkedimmunosorbent assays (ELISAs), Western blots, immunoprecipitations andimmunofluorescence using a detection reagent, such as an antibody orprotein binding agent. Alternatively, the peptide can be detected invivo in a subject by introducing into the subject a labeled anti-peptideantibody or other types of detection agent. For example, the antibodycan be labeled with a radioactive marker whose presence and location ina subject can be detected by standard imaging techniques. Particularlyuseful are methods that detect the allelic variant of a peptideexpressed in a subject and methods which detect fragments of a peptidein a sample.

The peptides are also useful in pharmacogenomic analysis.Pharmacogenomics deal with clinically significant hereditary variationsin the response to drugs due to altered drug disposition and abnormalaction in affected persons. See, e.g., Eichelbaum, M. (Clin. Exp.Pharmacol. Physiol. 23(10-11):983-985 (1996)), and Linder, M. W. (Clin.Chem. 43(2):254-266 (1997)). The clinical outcomes of these variationsresult in severe toxicity of therapeutic drugs in certain individuals ortherapeutic failure of drugs in certain individuals as a result ofindividual variation in metabolism. Thus, the genotype of the individualcan determine the way a therapeutic compound acts on the body or the waythe body metabolizes the compound. Further, the activity of drugmetabolizing enzymes effects both the intensity and duration of drugaction. Thus, the pharmacogenomics of the individual permit theselection of effective compounds and effective dosages of such compoundsfor prophylactic or therapeutic treatment based on the individual'sgenotype. The discovery of genetic polymorphisms in some drugmetabolizing enzymes has explained why some patients do not obtain theexpected drug effects, show an exaggerated drug effect, or experienceserious toxicity from standard drug dosages. Polymorphisms can beexpressed in the phenotype of the extensive metabolizer and thephenotype of the poor metabolizer. Accordingly, genetic polymorphism maylead to allelic protein variants of the phospholipase protein in whichone or more of the phospholipase functions in one population isdifferent from those in another population. The peptides thus allow atarget to ascertain a genetic predisposition that can affect treatmentmodality. Thus, in a ligand-based treatment, polymorphism may give riseto amino terminal extracellular domains and/or other substrate-bindingregions that are more or less active in substrate binding, andphospholipase activation. Accordingly, substrate dosage wouldnecessarily be modified to maximize the therapeutic effect within agiven population containing a polymorphism. As an alternative togenotyping, specific polymorphic peptides could be identified.

The peptides are also useful for treating a disorder characterized by anabsence of, inappropriate, or unwanted expression of the protein.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. Accordingly, methods fortreatment include the use of the phospholipase protein or fragments.

Antibodies

The invention also provides antibodies that selectively bind to one ofthe peptides of the present invention, a protein comprising such apeptide, as well as variants and fragments thereof. As used herein, anantibody selectively binds a target peptide when it binds the targetpeptide and does not significantly bind to unrelated proteins. Anantibody is still considered to selectively bind a peptide even if italso binds to other proteins that are not substantially homologous withthe target peptide so long as such proteins share homology with afragment or domain of the peptide target of the antibody. In this case,it would be understood that antibody binding to the peptide is stillselective despite some degree of cross-reactivity.

As used herein, an antibody is defined in terms consistent with thatrecognized within the art: they are multi-subunit proteins produced by amammalian organism in response to an antigen challenge. The antibodiesof the present invention include polyclonal antibodies and monoclonalantibodies, as well as fragments of such antibodies, including, but notlimited to, Fab or F(ab′), and Fv fragments.

Many methods are known for generating and/or identifying antibodies to agiven target peptide. Several such methods are described by Harlow,Antibodies, Cold Spring Harbor Press, (1989).

In general, to generate antibodies, an isolated peptide is used as animmunogen and is administered to a mammalian organism, such as a rat,rabbit or mouse. The full-length protein, an antigenic peptide fragmentor a fusion protein can be used. Particularly important fragments arethose covering fuinctional domains, such as the domains identified inFIG. 2, and domain of sequence homology or divergence amongst thefamily, such as those that can readily be identified using proteinalignment methods and as presented in the Figures.

Antibodies are preferably prepared from regions or discrete fragments ofthe phospholipase proteins. Antibodies can be prepared from any regionof the peptide as described herein. However, preferred regions willinclude those involved in function/activity and/or phospholipase/bindingpartner interaction. FIG. 2 can be used to identify particularlyimportant regions while sequence alignment can be used to identifyconserved and unique sequence fragments.

An antigenic fragment will typically comprise at least 8 contiguousamino acid residues. The antigenic peptide can comprise, however, atleast 10, 12, 14, 16 or more amino acid residues. Such fragments can beselected on a physical property, such as fragments correspond to regionsthat are located on the surface of the protein, e.g., hydrophilicregions or can be selected based on sequence uniqueness (see FIG. 2).

Detection on an antibody of the present invention can be facilitated bycoupling (i.e., physically linking) the antibody to a detectablesubstance. Examples of detectable substances include various enzymes,prosthetic groups, fluorescent materials, luminescent materials,bioluminescent materials, and radioactive materials. Examples ofsuitable enzymes include horseradish peroxidase, alkaline phosphatase,β-galactosidase, or acetylcholinesterase; examples of suitableprosthetic group complexes include streptavidinibiotin andavidinibiotin; examples of suitable fluorescent materials includeumbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine,dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; anexample of a luminescent material includes luminol; examples ofbioluminescent materials include luciferase, luciferin, and aequorin,and examples of suitable radioactive material include ¹²⁵I, ¹³¹I, ³⁵S or³H.

Antibody Uses

The antibodies can be used to isolate one of the proteins of the presentinvention by standard techniques, such as affinity chromatography orimmunoprecipitation. The antibodies can facilitate the purification ofthe natural protein from cells and recombinantly produced proteinexpressed in host cells. In addition, such antibodies are useful todetect the presence of one of the proteins of the present invention incells or tissues to determine the pattern of expression of the proteinamong various tissues in an organism and over the course of normaldevelopment. Experimental data as provided in FIG. 1 indicates thatphospholipase proteins of the present invention are expressed in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. Specifically, a virtual northernblot shows expression in the brain (including neuroblastomas and infantbrain), uterus, lung, and ovary adenocarcinomas. In addition, PCR-basedtissue screening panels indicate expression in leukocytes. Further, suchantibodies can be used to detect protein in situ, in vitro, or in a celllysate or supernatant in order to evaluate the abundance and pattern ofexpression. Also, such antibodies can be used to assess abnormal tissuedistribution or abnormal expression during development or progression ofa biological condition. Antibody detection of circulating fragments ofthe full length protein can be used to identify turnover.

Further, the antibodies can be used to assess expression in diseasestates such as in active stages of the disease or in an individual witha predisposition toward disease related to the protein's function. Whena disorder is caused by an inappropriate tissue distribution,developmental expression, level of expression of the protein, orexpressed/processed form, the antibody can be prepared against thenormal protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the brain (including neuroblastomas and infantbrain), uterus, lung, ovary adenocarcinomas, and leukocytes. If adisorder is characterized by a specific mutation in the protein,antibodies specific for this mutant protein can be used to assay for thepresence of the specific mutant protein.

The antibodies can also be used to assess normal and aberrantsubcellular localization of cells in the various tissues in an organism.Experimental data as provided in FIG. 1 indicates expression in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. The diagnostic uses can beapplied, not only in genetic testing, but also in monitoring a treatmentmodality. Accordingly, where treatment is ultimately aimed at correctingexpression level or the presence of aberrant sequence and aberranttissue distribution or developmental expression, antibodies directedagainst the protein or relevant fragments can be used to monitortherapeutic efficacy.

Additionally, antibodies are useful in pharmacogenomic analysis. Thus,antibodies prepared against polymorphic proteins can be used to identifyindividuals that require modified treatment modalities. The antibodiesare also useful as diagnostic tools as an immunological marker foraberrant protein analyzed by electrophoretic mobility, isoelectricpoint, tryptic peptide digest, and other physical assays known to thosein the art.

The antibodies are also useful for tissue typing. Experimental data asprovided in FIG. 1 indicates expression in humans in the brain(including neuroblastomas and infant brain), uterus, lung, ovaryadenocarcinomas, and leukocytes. Thus, where a specific protein has beencorrelated with expression in a specific tissue, antibodies that arespecific for this protein can be used to identify a tissue type.

The antibodies are also useful for inhibiting protein function, forexample, blocking the binding of the phospholipase peptide to a bindingpartner such as a substrate. These uses can also be applied in atherapeutic context in which treatment involves inhibiting the protein'sfunction. An antibody can be used, for example, to block binding, thusmodulating (agonizing or antagonizing) the peptides activity. Antibodiescan be prepared against specific fragments containing sites required forfunction or against intact protein that is associated with a cell orcell membrane. See FIG. 2 for structural information relating to theproteins of the present invention.

The invention also encompasses kits for using antibodies to detect thepresence of a protein in a biological sample. The kit can compriseantibodies such as a labeled or labelable antibody and a compound oragent for detecting protein in a biological sample; means for.determining the amount of protein in the sample; means for comparing theamount of protein in the sample with a standard; and instructions foruse. Such a kit can be supplied to detect a single protein or epitope orcan be configured to detect one of a multitude of epitopes, such as inan antibody detection array. Arrays are described in detail below fornuleic acid arrays and similar methods have been developed for antibodyarrays.

Nucleic Acid Molecules

The present invention further provides isolated nucleic acid moleculesthat encode a phospholipase peptide or protein of the present invention(cDNA, transcript and genomic sequence). Such nucleic acid moleculeswill consist of, consist essentially of, or comprise a nucleotidesequence that encodes one of the phospholipase peptides of the presentinvention, an allelic variant thereof, or an ortholog or paralogthereof.

As used herein, an “isolated” nucleic acid molecule is one that isseparated from other nucleic acid present in the natural source of thenucleic acid. Preferably, an “isolated” nucleic acid is free ofsequences which naturally flank the nucleic acid (i.e., sequenceslocated at the 5′ and 3′ ends of the nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived. However, there canbe some flanking nucleotide sequences, for example up to about 5KB, 4KB,3KB, 2KB, or 1KB or less, particularly contiguous peptide encodingsequences and peptide encoding sequences within the same gene butseparated by introns in the genomic sequence. The important point isthat the nucleic acid is isolated from remote and unimportant flankingsequences such that it can be subjected to the specific manipulationsdescribed herein such as recombinant expression, preparation of probesand primers, and other uses specific to the nucleic acid sequences.

Moreover, an “isolated” nucleic acid molecule, such as a transcript/cDNAmolecule, can be substantially free of other cellular material, orculture medium when produced by recombinant techniques, or chemicalprecursors or other chemicals when chemically synthesized. However, thenucleic acid molecule can be fused to other coding or regulatorysequences and still be considered isolated.

For example, recombinant DNA molecules contained in a vector areconsidered isolated. Further examples of isolated DNA molecules includerecombinant DNA molecules maintained in heterologous host cells orpurified (partially or substantially) DNA molecules in solution.Isolated RNA molecules include in vivo or in vitro RNA transcripts ofthe isolated DNA molecules of the present invention. Isolated nucleicacid molecules according to the present invention further include suchmolecules produced synthetically.

Accordingly, the present invention provides nucleic acid molecules thatconsist of the nucleotide sequence shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule consists of a nucleotide sequence when thenucleotide sequence is the complete nucleotide sequence of the nucleicacid molecule.

The present invention further provides nucleic acid molecules thatconsist essentially of the nucleotide sequence shown in FIG. 1 or 3 (SEQID NO: 1, transcript sequence and SEQ ID NO:3, genomic sequence), or anynucleic acid molecule that encodes the protein provided in FIG. 2, SEQID NO:2. A nucleic acid molecule consists essentially of a nucleotidesequence when such a nucleotide sequence is present with only a fewadditional nucleic acid residues in the final nucleic acid molecule.

The present invention ftuther provides nucleic acid molecules thatcomprise the nucleotide sequences shown in FIG. 1 or 3 (SEQ ID NO:1,transcript sequence and SEQ ID NO:3, genomic sequence), or any nucleicacid molecule that encodes the protein provided in FIG. 2, SEQ ID NO:2.A nucleic acid molecule comprises a nucleotide sequence when thenucleotide sequence is at least part of the final nucleotide sequence ofthe nucleic acid molecule. In such a fashion, the nucleic acid moleculecan be only the nucleotide sequence or have additional nucleic acidresidues, such as nucleic acid residues that are naturally associatedwith it or heterologous nucleotide sequences. Such a nucleic acidmolecule can have a few additional nucleotides or can comprises severalhundred or more additional nucleotides. A brief description of howvarious types of these nucleic acid molecules can be readilymade/isolated is provided below.

In FIGS. 1 and 3, both coding and non-coding sequences are provided.Because of the source of the present invention, humans genomic sequence(FIG. 3) and cDNA/transcript sequences (FIG. 1), the nucleic acidmolecules in the Figures will contain genomic intronic sequences, 5′ and3′ non-coding sequences, gene regulatory regions and non-codingintergenic sequences. In general such sequence features are either notedin FIGS. 1 and 3 or can readily be identified using computational toolsknown in the art. As discussed below, some of the non-coding regions,particularly gene regulatory elements such as promoters, are useful fora variety of purposes, e.g. control of heterologous gene expression,target for identifying gene activity modulating compounds, and areparticularly claimed as fragments of the genomic sequence providedherein.

The isolated nucleic acid molecules can encode the mature protein plusadditional amino or carboxyl-terminal amino acids, or amino acidsinterior to the mature peptide (when the mature form has more than onepeptide chain, for instance). Such sequences may play a role inprocessing of a protein from precursor to a mature form, facilitateprotein trafficking, prolong or shorten protein half-life or facilitatemanipulation of a protein for assay or production, among other things.As generally is the case in situ, the additional amino acids may beprocessed away from the mature protein by cellular enzymes.

As mentioned above, the isolated nucleic acid molecules include, but arenot limited to, the sequence encoding the phospholipase peptide alone,the sequence encoding the mature peptide and additional codingsequences, such as a leader or secretory sequence (e.g., a pre-pro orpro-protein sequence), the sequence encoding the mature peptide, with orwithout the additional coding sequences, plus additional non-codingsequences, for example introns and non-coding 5′ and 3′ sequences suchas transcribed but non-translated sequences that play a role intranscription, mRNA processing (including splicing and polyadenylationsignals), ribosome binding and stability of mRNA. In addition, thenucleic acid molecule may be fused to a marker sequence encoding, forexample, a peptide that facilitates purification.

Isolated nucleic acid molecules can be in the form of RNA, such as mRNA,or in the form DNA, including cDNA and genomic DNA obtained by cloningor produced by chemical synthetic techniques or by a combinationthereof. The nucleic acid, especially DNA, can be double-stranded orsingle-stranded. Single-stranded nucleic acid can be the coding strand(sense strand) or the non-coding strand (anti-sense strand).

The invention further provides nucleic acid molecules that encodefragments of the peptides of the present invention as well as nucleicacid molecules that encode obvious variants of the phospholipaseproteins of the present invention that are described above. Such nucleicacid molecules may be naturally occurring, such as allelic variants(same locus), paralogs (different locus), and orthologs (differentorganism), or may be constructed by recombinant DNA methods or bychemical synthesis. Such non-naturally occurring variants may be made bymutagenesis techniques, including those applied to nucleic acidmolecules, cells, or organisms. Accordingly, as discussed above, thevariants can contain nucleotide substitutions, deletions, inversions andinsertions. Variation can occur in either or both the coding andnon-coding regions. The variations can produce both conservative andnon-conservative amino acid substitutions.

The present invention fturther provides non-coding fragments of thenucleic acid molecules provided in FIGS. 1 and 3. Preferred non-codingfragments include, but are not limited to, promoter sequences, enhancersequences, gene modulating sequences and gene termination sequences.Such fragments are useful in controlling heterologous gene expressionand in developing screens to identify gene-modulating agents. A promotercan readily be identified as being 5′ to the ATG start site in thegenomic sequence provided in FIG. 3.

A fragment comprises a contiguous nucleotide sequence greater than 12 ormore nucleotides. Further, a fragment could at least 30, 40, 50, 100,250 or 500 nucleotides in length. The length of the fragment will bebased on its intended use. For example, the fragment can encode epitopebearing regions of the peptide, or can be useful as DNA probes andprimers. Such fragments can be isolated using the known nucleotidesequence to synthesize an oligonucleotide probe. A labeled probe canthen be used to screen a cDNA library, genomic DNA library, or mRNA toisolate nucleic acid corresponding to the coding region. Further,primers can be used in PCR reactions to clone specific regions of gene.

A probe/primer typically comprises substantially a purifiedoligonucleotide or oligonucleotide pair. The oligonucleotide typicallycomprises a region of nucleotide sequence that hybridizes understringent conditions to at least about 12, 20, 25, 40, 50 or moreconsecutive nucleotides.

Orthologs, homologs, and allelic variants can be identified usingmethods well known in the art. As described in the Peptide Section,these variants comprise a nucleotide sequence encoding a peptide that istypically 60-70%, 70-80%, 80-90%, and more typically at least about90-95% or more homologous to the nucleotide sequence shown in the Figuresheets or a fragment of this sequence. Such nucleic acid molecules canreadily be identified as being able to hybridize under moderate tostringent conditions, to the nucleotide sequence shown in the Figuresheets or a fragment of the sequence. Allelic variants can readily bedetermined by genetic locus of the encoding gene. The gene encoding thenovel phospholipase protein of the present invention is located on agenome component that has been mapped to human chromosome 2 (asindicated in FIG. 3), which is supported by multiple lines of evidence,such as STS and BAC map data.

FIG. 3 provides information on SNPs that have been found in the geneencoding the phospholipase proteins of the present invention. SNPs wereidentified at nine different nucleotide positions. SNPs outside the ORFand in introns may affect control/regulatory elements.

As used herein, the term “hybridizes under stringent conditions” isintended to describe conditions for hybridization and washing underwhich nucleotide sequences encoding a peptide at least 60-70% homologousto each other typically remain hybridized to each other. The conditionscan be such that sequences at least about 60%, at least about 70%, or atleast about 80% or more homologous to each other typically remainhybridized to each other. Such stringent conditions are known to thoseskilled in the art and can be found in Current Protocols in MolecularBiology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6. One example ofstringent hybridization conditions are hybridization in 6×sodiumchloride/sodium citrate (SSC) at about 45C, followed by one or morewashes in 0.2×SSC, 0.1% SDS at 50-65C. Examples of moderate to lowstringency hybridization conditions are well known in the art.

Nucleic Acid Molecule Uses

The nucleic acid molecules of the present invention are useful forprobes, primers, chemical intermediates, and in biological assays. Thenucleic acid molecules are useful as a hybridization probe for messengerRNA, transcript/cDNA and genomic DNA to isolate full-length cDNA andgenomic clones encoding the peptide described in FIG. 2 and to isolatecDNA and genomic clones that correspond to variants (alleles, orthologs,etc.) producing the same or related peptides shown in FIG. 2. Asillustrated in FIG. 3, SNPs were identified at nine different nucleotidepositions.

The probe can correspond to any sequence along the entire length of thenucleic acid molecules provided in the Figures. Accordingly, it could bederived from 5′ noncoding regions, the coding region, and 3′ noncodingregions. However, as discussed, fragments are not to be construed asencompassing fragments disclosed prior to the present invention.

The nucleic acid molecules are also useful as primers for PCR to amplifyany given region of a nucleic acid molecule and are useful to synthesizeantisense molecules of desired length and sequence.

The nucleic acid molecules are also useful for constructing recombinantvectors. Such vectors include expression vectors that express a portionof, or all of, the peptide sequences. Vectors also include insertionvectors, used to integrate into another nucleic acid molecule sequence,such as into the cellular genome, to alter in situ expression of a geneand/or gene product. For example, an endogenous coding sequence can bereplaced via homologous recombination with all or part of the codingregion containing one or more specifically introduced mutations.

The nucleic acid molecules are also useful for expressing antigenicportions of the proteins.

The nucleic acid molecules are also useful as probes for determining thechromosomal positions of the nucleic acid molecules by means of in situhybridization methods. The gene encoding the novel phospholipase proteinof the present invention is located on a genome component that has beenmapped to human chromosome 2 (as indicated in FIG. 3), which issupported by multiple lines of evidence, such as STS and BAC map data.

The nucleic acid molecules are also useful in making vectors containingthe gene regulatory regions of the nucleic acid molecules of the presentinvention.

The nucleic acid molecules are also useful for designing ribozymescorresponding to all, or a part, of the mRNA produced from the nucleicacid molecules described herein.

The nucleic acid molecules are also useful for making vectors thatexpress part, or all, of the peptides.

The nucleic acid molecules are also useful for constructing host cellsexpressing a part, or all, of the nucleic acid molecules and peptides.

The nucleic acid molecules are also useful for constructing transgenicanimals expressing all, or a part, of the nucleic acid molecules andpeptides.

The nucleic acid molecules are also useful as hybridization probes fordetermining the presence, level, form and distribution of nucleic acidexpression. Experimental data as provided in FIG. 1 indicates thatphospholipase proteins of the present invention are expressed in humansin the brain (including neuroblastomas and infant brain), uterus, lung,ovary adenocarcinomas, and leukocytes. Specifically, a virtual northernblot shows expression in the brain (including neuroblastomas and infantbrain), uterus, lung, and ovary adenocarcinomas. In addition, PCR-basedtissue screening panels indicate expression in leukocytes. Accordingly,the probes can be used to detect the presence of, or to determine levelsof, a specific nucleic acid molecule in cells, tissues, and inorganisms. The nucleic acid whose level is determined can be DNA or RNA.Accordingly, probes corresponding to the peptides described herein canbe used to assess expression and/or gene copy number in a given cell,tissue, or organism. These uses are relevant for diagnosis of disordersinvolving an increase or decrease in phospholipase protein expressionrelative to normal results.

In vitro techniques for detection of mRNA include Northernhybridizations and in situ hybridizations. In vitro techniques fordetecting DNA includes Southern hybridizations and in situhybridization.

Probes can be used as a part of a diagnostic test kit for identifyingcells or tissues that express a phospholipase protein, such as bymeasuring a level of a phospholipase-encoding nucleic acid in a sampleof cells from a subject e.g., mRNA or genomic DNA, or determining if aphospholipase gene has been mutated. Experimental data as provided inFIG. 1 indicates that phospholipase proteins of the present inventionare expressed in humans in the brain (including neuroblastomas andinfant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.Specifically, a virtual northern blot shows expression in the brain(including neuroblastomas and infant brain), uterus, lung, and ovaryadenocarcinomas. In addition, PCR-based tissue screening panels indicateexpression in leukocytes.

Nucleic acid expression assays are useful for drug screening to identifycompounds that modulate phospholipase nucleic acid expression.

The invention thus provides a method for identifying a compound that canbe used to treat a disorder associated with nucleic acid expression ofthe phospholipase gene, particularly biological and pathologicalprocesses that are mediated by the phospholipase in cells and tissuesthat express it. Experimental data as provided in FIG. 1 indicatesexpression in humans in the brain (including neuroblastomas and infantbrain), uterus, lung, ovary adenocarcinomas, and leukocytes. The methodtypically includes assaying the ability of the compound to modulate theexpression of the phospholipase nucleic acid and thus identifying acompound that can be used to treat a disorder characterized by undesiredphospholipase nucleic acid expression. The assays can be performed incell-based and cell-free systems. Cell-based assays include cellsnaturally expressing the phospholipase nucleic acid or recombinant cellsgenetically engineered to express specific nucleic acid sequences.

The assay for phospholipase nucleic acid expression can involve directassay of nucleic acid levels, such as mRNA levels, or on collateralcompounds involved in the signal pathway. Further, the expression ofgenes that are up- or down-regulated in response to the phospholipaseprotein signal pathway can also be assayed. In this embodiment theregulatory regions of these genes can be operably linked to a reportergene such as luciferase.

Thus, modulators of phospholipase gene expression can be identified in amethod wherein a cell is contacted with a candidate compound and theexpression of mRNA determined. The level of expression of phospholipasemRNA in the presence of the candidate compound is compared to the levelof expression of phospholipase mRNA in the absence of the candidatecompound. The candidate compound can then be identified as a modulatorof nucleic acid expression based on this comparison and be used, forexample to treat a disorder characterized by aberrant nucleic acidexpression. When expression of mRNA is statistically significantlygreater in the presence of the candidate compound than in its absence,the candidate compound is identified as a stimulator of nucleic acidexpression. When nucleic acid expression is statistically significantlyless in the presence of the candidate compound than in its absence, thecandidate compound is identified as an inhibitor of nucleic acidexpression.

The invention further provides methods of treatment, with the nucleicacid as a target, using a compound identified through drug screening asa gene modulator to modulate phospholipase nucleic acid expression incells and tissues that express the phospholipase. Experimental data asprovided in FIG. 1 indicates that phospholipase proteins of the presentinvention are expressed in humans in the brain (including neuroblastomasand infant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.Specifically, a virtual northern blot shows expression in the brain(including neuroblastomas and infant brain), uterus, lung, and ovaryadenocarcinomas. In addition, PCR-based tissue screening panels indicateexpression in leukocytes. Modulation includes both up-regulation (i.e.activation or agonization) or down-regulation (suppression orantagonization) or nucleic acid expression.

Alternatively, a modulator for phospholipase nucleic acid expression canbe a small molecule or drug identified using the screening assaysdescribed herein as long as the drug or small molecule inhibits thephospholipase nucleic acid expression in the cells and tissues thatexpress the protein. Experimental data as provided in FIG. 1 indicatesexpression in humans in the brain (including neuroblastomas and infantbrain), uterus, lung, ovary adenocarcinomas, and leukocytes.

The nucleic acid molecules are also useful for monitoring theeffectiveness of modulating compounds on the expression or activity ofthe phospholipase gene in clinical trials or in a treatment regimen.Thus, the gene expression pattern can serve as a barometer for thecontinuing effectiveness of treatment with the compound, particularlywith compounds to which a patient can develop resistance. The geneexpression pattern can also serve as a marker indicative of aphysiological response of the affected cells to the compound.Accordingly, such monitoring would allow either increased administrationof the compound or the administration of alternative compounds to whichthe patient has not become resistant. Similarly, if the level of nucleicacid expression falls below a desirable level, administration of thecompound could be commensurately decreased.

The nucleic acid molecules are also useful in diagnostic assays forqualitative changes in phospholipase nucleic acid expression, andparticularly in qualitative changes that lead to pathology. The nucleicacid molecules can be used to detect mutations in phospholipase genesand gene expression products such as mRNA. The nucleic acid moleculescan be used as hybridization probes to detect naturally occurringgenetic mutations in the phospholipase gene and thereby to determinewhether a subject with the mutation is at risk for a disorder caused bythe mutation. Mutations include deletion, addition, or substitution ofone or more nucleotides in the gene, chromosomal rearrangement, such asinversion or transposition, modification of genomic DNA, such asaberrant methylation patterns or changes in gene copy number, such asamplification. Detection of a mutated form of the phospholipase geneassociated with a dysfunction provides a diagnostic tool for an activedisease or susceptibility to disease when the disease results fromoverexpression, underexpression, or altered expression of aphospholipase protein.

Individuals carrying mutations in the phospholipase gene can be detectedat the nucleic acid level by a variety of techniques. FIG. 3 providesinformation on SNPs that have been found in the gene encoding thephospholipase proteins of the present invention. SNPs were identified atnine different nucleotide positions. SNPs outside the ORF and in intronsmay affect control/regulatory elements. The gene encoding the novelphospholipase protein of the present invention is located on a genomecomponent that has been mapped to human chromosome 2 (as indicated inFIG. 3), which is supported by multiple lines of evidence, such as STSand BAC map data. Genomic DNA can be analyzed directly or can beamplified by using PCR prior to analysis. RNA or cDNA can be used in thesame way. In some uses, detection of the mutation involves the use of aprobe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S. Pat.Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegranet al., Science 241:1077-1080 (1988); and Nakazawa et al., PNAS91:360-364 (1994)), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al., NucleicAcids Res. 23:675-682 (1995)). This method can include the steps ofcollecting a sample of cells from a patient, isolating nucleic acid(e.g., genomic, mRNA or both) from the cells of the sample, contactingthe nucleic acid sample with one or more primers which specificallyhybridize to a gene under conditions such that hybridization andamplification of the gene (if present) occurs, and detecting thepresence or absence of an amplification product, or detecting the sizeof the amplification product and comparing the length to a controlsample. Deletions and insertions can be detected by a change in size ofthe amplified product compared to the normal genotype. Point mutationscan be identified by hybridizing amplified DNA to normal RNA orantisense DNA sequences.

Alternatively, mutations in a phospholipase gene can be directlyidentified, for example, by alterations in restriction enzyme digestionpatterns determined by gel electrophoresis.

Further, sequence-specific ribozymes (U.S. Pat. No. 5,498,531) can beused to score for the presence of specific mutations by development orloss of a ribozyme cleavage site. Perfectly matched sequences can bedistinguished from mismatched sequences by nuclease cleavage digestionassays or by differences in melting temperature.

Sequence changes at specific locations can also be assessed by nucleaseprotection assays such as RNase and S1 protection or the chemicalcleavage method. Furthermore, sequence differences between a mutantphospholipase gene and a wild-type gene can be determined by direct DNAsequencing. A variety of automated sequencing procedures can be utilizedwhen performing the diagnostic assays (Naeve, C. W., (1995)Biotechniques 19:448), including sequencing by mass spectrometry (see,e.g., PCT International Publication No. WO 94/16101; Cohen et al, Adv.Chromatogr. 36:127-162 (1996); and Griffin et al., Appl. Biochem.Biotechnol. 38:147-159 (1993)).

Other methods for detecting mutations in the gene include methods inwhich protection from cleavage agents is used to detect mismatched basesin RNA/RNA or RNA/DNA duplexes (Myers etal., Science 230:1242 (1985));Cotton et al., PNAS85:4397 (1988); Saleebaetal., Meth. Enzymol.217:286-295 (1992)), electrophoretic mobility of mutant and wild typenucleic acid is compared (Orita et al., PNAS 86:2766 (1989); Cotton etal., Mutat. Res. 285:125-144 (1993); and Hayashi et al., Genet. Anal.Tech. Appl. 9:73-79 (1992)), and movement of mutant or wild-typefragments in polyacrylamide gels containing a gradient of denaturant isassayed using denaturing gradient gel electrophoresis (Myers et al.,Nature 313:495 (1985)). Examples of other techniques for detecting pointmutations include selective oligonucleotide hybridization, selectiveamplification, and selective primer extension.

The nucleic acid molecules are also useful for testing an individual fora genotype that while not necessarily causing the disease, neverthelessaffects the treatment modality. Thus, the nucleic acid molecules can beused to study the relationship between an individual's genotype and theindividual's response to a compound used for treatment (pharmacogenomicrelationship). Accordingly, the nucleic acid molecules described hereincan be used to assess the mutation content of the phospholipase gene inan individual in order to select an appropriate compound or dosageregimen for treatment. FIG. 3 provides information on SNPs that havebeen found in the gene encoding the phospholipase proteins of thepresent invention. SNPs were identified at nine different nucleotidepositions. SNPs outside the ORF and in introns may affectcontrol/regulatory elements.

Thus nucleic acid molecules displaying genetic variations that affecttreatment provide a diagnostic target that can be used to tailortreatment in an individual. Accordingly, the production of recombinantcells and animals containing these polymorphisms allow effectiveclinical design of treatment compounds and dosage regimens.

The nucleic acid molecules are thus useful as antisense constructs tocontrol phospholipase gene expression in cells, tissues, and organisms.A DNA antisense nucleic acid molecule is designed to be complementary toa region of the gene involved in transcription, preventing transcriptionand hence production of phospholipase protein. An antisense RNA or DNAnucleic acid molecule would hybridize to the mRNA and thus blocktranslation of mRNA into phospholipase protein.

Alternatively, a class of antisense molecules can be used to inactivatemRNA in order to decrease expression of phospholipase nucleic acid.Accordingly, these molecules can treat a disorder characterized byabnormal or undesired phospholipase nucleic acid expression. Thistechnique involves cleavage by means of ribozymes containing nucleotidesequences complementary to one or more regions in the MRNA thatattenuate the ability of the mRNA to be translated. Possible regionsinclude coding regions and particularly coding regions corresponding tothe catalytic and other functional activities of the phospholipaseprotein, such as substrate binding.

The nucleic acid molecules also provide vectors for gene therapy inpatients containing cells that are aberrant in phospholipase geneexpression. Thus, recombinant cells, which include the patient's cellsthat have been engineered ex vivo and returned to the patient, areintroduced into an individual where the cells produce the desiredphospholipase protein to treat the individual.

The invention also encompasses kits for detecting the presence of aphospholipase nucleic acid in a biological sample. Experimental data asprovided in FIG. 1 indicates that phospholipase proteins of the presentinvention are expressed in humans in the brain (including neuroblastomasand infant brain), uterus, lung, ovary adenocarcinomas, and leukocytes.Specifically, a virtual northern blot shows expression in the brain(including neuroblastomas and infant brain), uterus, lung, and ovaryadenocarcinomas. In addition, PCR-based tissue screening panels indicateexpression in leukocytes. For example, the kit can comprise reagentssuch as a labeled or labelable nucleic acid or agent capable ofdetecting phospholipase nucleic acid in a biological sample; means fordetermining the amount of phospholipase nucleic acid in the sample; andmeans for comparing the amount of phospholipase nucleic acid in thesample with a standard. The compound or agent As can be packaged in asuitable container. The kit can further comprise instructions for usingthe kit to detect phospholipase protein mRNA or DNA.

Nucleic Acid Arrays

The present invention further provides nucleic acid detection kits, suchas arrays or microarrays of nucleic acid molecules that are based on thesequence information provided in FIGS. 1 and 3 (SEQ ID NOS:1 and 3).

As used herein “Arrays” or “Microarrays” refers to an array of distinctpolynucleotides or oligonucleotides synthesized on a substrate, such aspaper, nylon or other type of membrane, filter, chip, glass slide, orany other suitable solid support. In one embodiment, the microarray isprepared and used according to the methods described in U.S. Pat. No.5,837,832, Chee et al., PCT application WO95/11995 (Chee et al.),Lockhart, D. J. et al. (1996; Nat. Biotech. 14: 1675-1680) and Schena,M. et al (1996; Proc. Natl. Acad. Sci. 93: 10614-10619), all of whichare incorporated herein in their entirety by reference. In otherembodiments, such arrays are produced by the methods described by Brownet al., U.S. Pat. No. 5,807,522.

The microarray or detection kit is preferably composed of a large numberof unique, single-stranded nucleic acid sequences, usually eithersynthetic antisense oligonucleotides or fragments of cDNAs, fixed to asolid support. The oligonucleotides are preferably about 6-60nucleotides in length, more preferably 15-30 nucleotides in length, andmost preferably about 20-25 nucleotides in length. For a certain type ofmicroarray or detection kit, it may be preferable to useoligonucleotides that are only 7-20 nucleotides in length. Themicroarray or detection kit may contain oligonucleotides that cover theknown 5′, or 3′, sequence, sequential oligonucleotides which cover thefull length sequence; or unique oligonucleotides selected fromparticular areas along the length of the sequence. Polynucleotides usedin the microarray or detection kit may be oligonucleotides that arespecific to a gene or genes of interest.

In order to produce oligonucleotides to a known sequence for amicroarray or detection kit, the gene(s) of interest (or an ORFidentified from the contigs of the present invention) is typicallyexamined using a computer algorithm which starts at the 5′ or at the 3′end of the nucleotide sequence. Typical algorithms will then identifyoligomers of defined length that are unique to the gene, have a GCcontent within a range suitable for hybridization, and lack predictedsecondary structure that may interfere with hybridization. In certainsituations it may be appropriate to use pairs of oligonucleotides on amicroarray or detection kit. The “pairs” will be identical, except forone nucleotide that preferably is located in the center of the sequence.The second oligonucleotide in the pair (mismatched by one) serves as acontrol. The number of oligonucleotide pairs may range from two to onemillion. The oligomers are synthesized at designated areas on asubstrate using a light-directed chemical process. The substrate may bepaper, nylon or other type of membrane, filter, chip, glass slide or anyother suitable solid support.

In another aspect, an oligonucleotide may be synthesized on the surfaceof the substrate by using a chemical coupling procedure and an ink jetapplication apparatus, as described in PCT application WO95/25 1116(Baldeschweiler et al.) which is incorporated herein in its entirety byreference. In another aspect, a “gridded” array analogous to a dot (orslot) blot may be used to arrange and link cDNA fragments oroligonucleotides to the surface of a substrate using a vacuum system,thermal, UV, mechanical or chemical bonding procedures. An array, suchas those described above, may be produced by hand or by using availabledevices (slot blot or dot blot apparatus), materials (any suitable solidsupport), and machines (including robotic instruments), and may contain8, 24, 96, 384, 1536, 6144 or more oligonucleotides, or any other numberbetween two and one million which lends itself to the efficient use ofcommercially available instrumentation.

In order to conduct sample analysis using a microarray or detection kit,the RNA or DNA from a biological sample is made into hybridizationprobes. The mRNA is isolated, and cDNA is produced and used as atemplate to make antisense RNA (aRNA). The aRNA is amplified in thepresence of fluorescent nucleotides, and labeled probes are incubatedwith the microarray or detection kit so that the probe sequenceshybridize to complementary oligonucleotides of the microarray ordetection kit. Incubation conditions are adjusted so that hybridizationoccurs with precise complementary matches or with various degrees ofless complementarity. After removal of nonhybridized probes, a scanneris used to determine the levels and patterns of fluorescence. Thescanned images are examined to determine degree of complementarity andthe relative abundance of each oligonucleotide sequence on themicroarray or detection kit. The biological samples may be obtained fromany bodily fluids (such as blood, urine, saliva, phlegm, gastric juices,etc.), cultured cells, biopsies, or other tissue preparations. Adetection system may be used to measure the absence, presence, andamount of hybridization for all of the distinct sequencessimultaneously. This data may be used for large-scale correlationstudies on the sequences, expression patterns, mutations, variants, orpolymorphisms among samples.

Using such arrays, the present invention provides methods to identifythe expression of the phospholipase proteins/peptides of the presentinvention. In detail, such methods comprise incubating a test samplewith one or more nucleic acid molecules and assaying for binding of thenucleic acid molecule with components within the test sample. Suchassays will typically involve arrays comprising many genes, at least oneof which is a gene of the present invention and or alleles of thephospholipase gene of the present invention. FIG. 3 provides informationon SNPs that have been found in the gene encoding the phospholipaseproteins of the present invention. SNPs were identified at ninedifferent nucleotide positions. SNPs outside the ORF and in introns mayaffect control/regulatory elements.

Conditions for incubating a nucleic acid molecule with a test samplevary. Incubation conditions depend on the format employed in the assay,the detection methods employed, and the type and nature of the nucleicacid molecule used in the assay. One skilled in the art will recognizethat any one of the commonly available hybridization, amplification orarray assay formats can readily be adapted to employ the novel fragmentsof the Human genome disclosed herein. Examples of such assays can befound in Chard, T, An Introduction to Radioimmunoassay and RelatedTechniques, Elsevier Science Publishers, Amsterdam, The Netherlands(1986); Bullock, G. R. et al., Techniques in Immunocytochemistry,Academic Press, Orlando, Fla. Vol. 1 (1982), Vol. 2 (1983), Vol. 3(1985); Tijssen, P., Practice and Theory ofEnzyme Immunoassays:Laboratory Techniques in Biochemistry and Molecular Biology, ElsevierScience Publishers, Amsterdam, The Netherlands (1985).

The test samples of the present invention include cells; protein ormembrane extracts of cells. The test sample used in the above-describedmethod will vary based on the assay format, nature of the detectionmethod and the tissues, cells or extracts used as the sample to beassayed. Methods for preparing nucleic acid extracts or of cells arewell known in the art and can be readily be adapted in order to obtain asample that is compatible with the system utilized.

In another embodiment of the present invention, kits are provided whichcontain the necessary reagents to carry out the assays of the presentinvention.

Specifically, the invention provides a compartmentalized kit to receive,in close confinement, one or more containers which comprises: (a) afirst container comprising one of the nucleic acid molecules that canbind to a fragment of the Human genome disclosed herein; and (b) one ormore other containers comprising one or more of the following: washreagents, reagents capable of detecting presence of a bound nucleicacid.

In detail, a compartmentalized kit includes any kit in which reagentsare contained in separate containers. Such containers include smallglass containers, plastic containers, strips of plastic, glass or paper,or arraying material such as silica. Such containers allows one toefficiently transfer reagents from one compartment to anothercompartment such that the samples and reagents are notcross-contaminated, and the agents or solutions of each container can beadded in a quantitative fashion from one compartment to another. Suchcontainers will include a container which will accept the test sample, acontainer which contains the nucleic acid probe, containers whichcontain wash reagents (such as phosphate buffered saline, Tris-buffers,etc.), and containers which contain the reagents used to detect thebound probe. One skilled in the art will readily recognize that thepreviously unidentified phospholipase gene of the present invention canbe routinely identified using the sequence information disclosed hereincan be readily incorporated into one of the established kit formatswhich are well known in the art, particularly expression arrays.

Vectors/host Cells

The invention also provides vectors containing the nucleic acidmolecules described herein. The term “vector” refers to a vehicle,preferably a nucleic acid molecule, which can transport the nucleic acidmolecules. When the vector is a nucleic acid molecule, the nucleic acidmolecules are covalently linked to the vector nucleic acid. With thisaspect of the invention, the vector includes a plasmid, single or doublestranded phage, a single or double stranded RNA or DNA viral vector, orartificial chromosome, such as a BAC, PAC, YAC, OR MAC.

A vector can be maintained in the host cell as an extrachromosomalelement where it replicates and produces additional copies of thenucleic acid molecules. Alternatively, the vector may integrate into thehost cell genome and produce additional copies of the nucleic acidmolecules when the host cell replicates.

The invention provides vectors for the maintenance (cloning vectors) orvectors for expression (expression vectors) of the nucleic acidmolecules. The vectors can function in prokaryotic or eukaryotic cellsor in both (shuttle vectors).

Expression vectors contain cis-acting regulatory regions that areoperably linked in the vector to the nucleic acid molecules such thattranscription of the nucleic acid molecules is allowed in a host cell.The nucleic acid molecules can be introduced into the host cell with aseparate nucleic acid molecule capable of affecting transcription. Thus,the second nucleic acid molecule may provide a trans-acting factorinteracting with the cis-regulatory control region to allowtranscription of the nucleic acid. molecules from the vector.Alternatively, a trans-acting factor may be supplied by the host cell.Finally, a trans-acting factor can be produced from the vector itself.It is understood, however, that in some embodiments, transcriptionand/or translation of the nucleic acid molecules can occur in acell-free system.

The regulatory sequence to which the nucleic acid molecules describedherein can be operably linked include promoters for directing mRNAtranscription. These include, but are not limited to, the left promoterfrom bacteriophage X, the lac, TRP, and TAC promoters from E. coli, theearly and late promoters from SV40, the CMV immediate early promoter,the adenovirus early and late promoters, and retrovirus long-terninalrepeats.

In addition to control regions that promote transcription, expressionvectors may also include regions that modulate transcription, such asrepressor binding sites and enhancers. Examples include the SV40enhancer, the cytomegalovirus immediate early enhancer, polyomaenhancer, adenovirus enhancers, and retrovirus LTR enhancers.

In addition to containing sites for transcription initiation andcontrol, expression vectors can also contain sequences necessary fortranscription termination and, in the transcribed region a ribosomebinding site for translation. Other regulatory control elements forexpression include initiation and termination codons as well aspolyadenylation signals. The person of ordinary skill in the art wouldbe aware of the numerous regulatory sequences that are useful inexpression vectors. Such regulatory sequences are described, forexample, in Sambrook et al., Molecular Cloning: A Laboratory Manual. 2nded., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,(1989).

A variety of expression vectors can be used to express a nucleic acidmolecule. Such vectors include chromosomal, episomal, and virus-derivedvectors, for example vectors derived from bacterial plasmids, frombacteriophage, from yeast episomes, from yeast chromosomal elements,including yeast artificial chromosomes, from viruses such asbaculoviruses, papovaviruses such as SV40, Vaccinia viruses,adenoviruses, poxviruses, pseudorabies viruses, and retroviruses.Vectors may also be derived from combinations of these sources such asthose derived from plasmid and bacteriophage genetic elements, e.g.cosmids and phagemids. Appropriate cloning and expression vectors forprokaryotic and eukaryotic hosts are described in Sambrook et al.,Molecular Cloning: A Laboratory Manual. 2nd ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (1989).

The regulatory sequence may provide constitutive expression in one ormore host cells (i.e. tissue specific) or may provide for inducibleexpression in one or more cell types such as by temperature, nutrientadditive, or exogenous factor such as a hormone or other ligand. Avariety of vectors providing for constitutive and inducible expressionin prokaryotic and eukaryotic hosts are well known to those of ordinaryskill in the art.

The nucleic acid molecules can be inserted into the vector nucleic acidby well-known methodology. Generally, the DNA sequence that willultimately be expressed is joined to an expression vector by cleavingthe DNA sequence and the expression vector with one or more restrictionenzymes and then ligating the fragments together. Procedures forrestriction enzyme digestion and ligation are well known to those ofordinary skill in the art.

The vector containing the appropriate nucleic acid molecule can beintroduced into an appropriate host cell for propagation or expressionusing well-known techniques. Bacterial cells include, but are notlimited to, E. coli, Streptomyces, and Salmonella typhimurium.Eukaryotic cells include, but are not limited to, yeast, insect cellssuch as Drosophila, animal cells such as COS and CHO cells, and plantcells.

As described herein, it may be desirable to express the peptide as afusion protein. Accordingly, the invention provides fusion vectors thatallow for the production of the peptides. Fusion vectors can increasethe expression of a recombinant protein, increase the solubility of therecombinant protein, and aid in the purification of the protein byacting for example as a ligand for affinity purification. A proteolyticcleavage site may be introduced at the junction of the fusion moiety sothat the desired peptide can ultimately be separated from the fusionmoiety. Proteolytic enzymes include, but are not limited to, factor Xa,thrombin, and enterophospholipase. Typical fusion expression vectorsinclude pGEX (Smith et al., Gene 67:31-40 (1988)), pMAL (New EnglandBiolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) whichfuse glutathione S-transferase (GST), maltose E binding protein, orprotein A, respectively, to the target recombinant protein. Examples ofsuitable inducible non-fusion E. coli expression vectors include pTrc(Amarn et al., Gene 69:301-315 (1988)) and pET I Id (Studier et al.,Gene Expression Technology:

Methods in Enzymology 185:60-89 (1990)). Recombinant protein expressioncan be maximized in host bacteria by providing a genetic backgroundwherein the host cell has an impaired capacity to proteolytically cleavethe recombinant protein. (Gottesman, S., Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990)119-128). Alternatively, the sequence of the nucleic acid molecule ofinterest can be altered to provide preferential codon usage for aspecific host cell, for example E. coli. (Wada et al., Nucleic AcidsRes. 20:2111-2118 (1992)).

The nucleic acid molecules can also be expressed by expression vectorsthat are operative in yeast. Examples of vectors for expression in yeaste.g., S. cerevisiae include pYepSec1 (Baldari, et al., EMBO J. 6:229-234(1987)), pMFa (Kujan et al., Cell 30:933-943(1982)), pJRY88 (Schultz etal., Gene 54:113-123 (1987)), and pYES2 (Invitrogen Corporation, SanDiego, Calif.).

The nucleic acid molecules can also be expressed in insect cells using,for example, baculovirus expression vectors. Baculovirus vectorsavailable for expression of proteins in cultured insect cells (e.g., Sf9 cells) include the pAc series (Smith et al., Mol. Cell Biol.3:2156-2165 (1983)) and the pVL series (Lucklow et al., Virology170:31-39 (1989)).

In certain embodiments of the invention, the nucleic acid moleculesdescribed herein are expressed in mammalian cells using mammalianexpression vectors. Examples of mammalian expression vectors includepCDM8 (Seed, B. Nature 329:840(1987)) and pMT2PC (Kaufman et al., EMBOJ. 6:187-195 (1987)).

The expression vectors listed herein are provided by way of example onlyof the well-known vectors available to those of ordinary skill in theart that would be useful to express the nucleic acid molecules. Theperson of ordinary skill in the art would be aware of other vectorssuitable for maintenance propagation or expression of the nucleic acidmolecules described herein. These are found for example in Sambrook, J.,Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual.2nd, ed, Cold Spring Harbor Laboratory, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 1989.

The invention also encompasses vectors in which the nucleic acidsequences described herein are cloned into the vector in reverseorientation, but operably linked to a regulatory sequence that permitstranscription of antisense RNA. Thus, an antisense transcript can beproduced to all, or to a portion, of the nucleic acid molecule sequencesdescribed herein, including both coding and non-coding regions.Expression of this antisense RNA is subject to each of the parametersdescribed above in relation to expression of the sense RNA (regulatorysequences, constitutive or inducible expression, tissue-specificexpression).

The invention also relates to recombinant host cells containing thevectors described herein. Host cells therefore include prokaryoticcells, lower eukaryotic cells such as yeast, other eukaryotic cells suchas insect cells, and higher eukaryotic cells such as mammalian cells.

The recombinant host cells are prepared by introducing the vectorconstructs described herein into the cells by techniques readilyavailable to the person of ordinary skill in the art. These include, butare not limited to, calcium phosphate transfection,DEAE-dextran-mediated transfection, cationic lipid-mediatedtransfection, electroporation, transduction, infection, lipofection, andother techniques such as those found in Sambrook, et al. (MolecularCloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).

Host cells can contain more than one vector. Thus, different nucleotidesequences can be introduced on different vectors of the same cell.Similarly, the nucleic acid molecules can be introduced either alone orwith other nucleic acid molecules that are not related to the nucleicacid molecules such as those providing trans-acting factors forexpression vectors. When more than one vector is introduced into a cell,the vectors can be introduced independently, co-introduced or joined tothe nucleic acid molecule vector.

In the case of bacteriophage and viral vectors, these can be introducedinto cells as packaged or encapsulated virus by standard procedures forinfection and transduction. Viral vectors can be replication-competentor replication-defective. In the case in which viral replication isdefective, replication will occur in host cells providing functions thatcomplement the defects.

Vectors generally include selectable markers that enable the selectionof the subpopulation of cells that contain the recombinant vectorconstructs. The marker can be contained in the same vector that containsthe nucleic acid molecules described herein or may be on a separatevector. Markers include tetracycline or ampicillin-resistance genes forprokaryotic host cells and dihydrofolate reductase or neomycinresistance for eukaryotic host cells. However, any marker that providesselection for a phenotypic trait will be effective.

While the mature proteins can be produced in bacteria, yeast, mammaliancells, and other cells under the control of the appropriate regulatorysequences, cell- free transcription and translation systems can also beused to produce these proteins using RNA derived from the DNA constructsdescribed herein.

Where secretion of the peptide is desired, which is difficult to achievewith multi-transmembrane domain containing proteins such asphospholipases, appropriate secretion signals are incorporated into thevector. The signal sequence can be endogenous to the peptides orheterologous to these peptides.

Where the peptide is not secreted into the medium, which is typicallythe case with phospholipases, the protein can be isolated from the hostcell by standard disruption procedures, including freeze thaw,sonication, mechanical disruption, use of lysing agents and the like.The peptide can then be recovered and purified by well-knownpurification methods including ammonium sulfate precipitation, acidextraction, anion or cationic exchange chromatography, phosphocellulosechromatography, hydrophobic-interaction chromatography, affinitychromatography, hydroxylapatite chromatography, lectin chromatography,or high performance liquid chromatography.

It is also understood that depending upon the host cell in recombinantproduction of the peptides described herein, the peptides can havevarious glycosylation patterns, depending upon the cell, or maybenon-glycosylated as when produced in bacteria. In addition, the peptidesmay include an initial modified methionine in some cases as a result ofa host-mediated process.

Uses of Vectors and Host Cells

The recombinant host cells expressing the peptides described herein havea variety of uses. First, the cells are useful for producing aphospholipase protein or peptide that can be further purified to producedesired amounts of phospholipase protein or fragments. Thus, host cellscontaining expression vectors are useful for peptide production.

Host cells are also useful for conducting cell-based assays involvingthe phospholipase protein or phospholipase protein fragments, such asthose described above as well as other formats known in the art. Thus, arecombinant host cell expressing a native phospholipase protein isuseful for assaying compounds that stimulate or inhibit phospholipaseprotein function.

Host cells are also useful for identifying phospholipase protein mutantsin which these functions are affected. If the mutants naturally occurand give rise to a pathology, host cells containing the mutations areuseful to assay compounds that have a desired effect on the mutantphospholipase protein (for example, stimulating or inhibiting function)which may not be indicated by their effect on the native phospholipaseprotein.

Genetically engineered host cells can be further used to producenon-human transgenic animals. A transgenic animal is preferably amammal, for example a rodent, such as a rat or mouse, in which one ormore of the cells of the animal include a transgene. A transgene isexogenous DNA which is integrated into the genome of a cell from which atransgenic animal develops and which remains in the genome of the matureanimal in one or more cell types or tissues of the transgenic animal.These animals are useful for studying the function of a phospholipaseprotein and identifying and evaluating modulators of phospholipaseprotein activity. Other examples of transgenic animals include non-humanprimates, sheep, dogs, cows, goats, chickens, and amphibians.

A transgenic animal can be produced by introducing nucleic acid into themale pronuclei of a fertilized oocyte, e.g., by microinjection,retroviral infection, and allowing the oocyte to develop in apseudopregnant female foster animal. Any of the phospholipase proteinnucleotide sequences can be introduced as a transgene into the genome ofa non-human animal, such as a mouse.

Any of the regulatory or other sequences useful in expression vectorscan form part of the transgenic sequence. This includes intronicsequences and polyadenylation signals, if not already included. Atissue-specific regulatory sequence(s) can be operably linked to thetransgene to direct expression of the phospholipase protein toparticular cells.

Methods for generating transgenic animals via embryo manipulation andmicroinjection, particularly animals such as mice, have becomeconventional in the art and are described, for example, in U.S. Pat.Nos. 4,736,866 and 4,870,009, both by Leder et al, U.S. Pat. No.4,873,191 by Wagner et al. and in Hogan, B., Manipulating the MouseEmbryo, (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,1986). Similar methods are used for production of other transgenicanimals. A transgenic founder animal can be identified based upon thepresence of the transgene in its genome and/or expression of transgenicmRNA in tissues or cells of the animals. A transgenic founder animal canthen be used to breed additional animals carrying the transgene.Moreover, transgenic animals carrying a transgene can further be bred toother transgenic animals carrying other transgenes. A transgenic animalalso includes animals in which the entire animal or tissues in theanimal have been produced using the homologously recombinant host cellsdescribed herein.

In another embodiment, transgenic non-human animals can be producedwhich contain selected systems that allow for regulated expression ofthe transgene. One example of such a system is the cre/loxP recombinasesystem of bacteriophage P 1. For a description of the cre/loxPrecombinase system, see, e.g., Lakso et al. PNAS 89:6232-6236 (1992).Another example of a recombinase system is the FLP recombinase system ofS. cerevisiae (O'Gorman et al. Science 251:1351-1355 (1991). If acre/loxP recombinase system is used to regulate expression of thetransgene, animals containing transgenes encoding both the Crerecombinase and a selected protein is required. Such animals can beprovided through the construction of “double” transgenic animals, e.g.,by mating two transgenic animals, one containing a transgene encoding aselected protein and the other containing a transgene encoding arecombinase.

Clones of the non-human transgenic animals described herein can also beproduced according to the methods described in Wilmut, I. et al. Nature385:810-813 (1997) and PCT International Publication Nos. WO 97/07668and WO 97/07669. In brief, a cell, e.g., a somatic cell, from thetransgenic animal can be isolated and induced to exit the growth cycleand enter G_(o) phase. The quiescent cell can then be fused, e.g.,through the use of electrical pulses, to an enucleated oocyte from ananimal of the same species from which the quiescent cell is isolated.The reconstructed oocyte is then cultured such that it develops tomorula or blastocyst and then transferred to pseudopregnant femalefoster animal. The offspring born of this female foster animal will be aclone of the animal from which the cell, e.g., the somatic cell, isisolated.

Transgenic animals containing recombinant cells that express thepeptides described herein are useful to conduct the assays describedherein in an in vivo context. Accordingly, the various physiologicalfactors that are present in vivo and that could effect substratebinding, phospholipase protein activation, and signal transduction, maynot be evident from in vitro cell-free or cell-based assays.Accordingly, it is useful to provide non-human transgenic animals toassay in vivo phospholipase protein function, including substrateinteraction, the effect of specific mutant phospholipase proteins onphospholipase protein function and substrate interaction, and the effectof chimeric phospholipase proteins. It is also possible to assess theeffect of null mutations, that is, mutations that substantially orcompletely eliminate one or more phospholipase protein functions.

All publications and patents mentioned in the above specification areherein incorporated by reference. Various modifications and variationsof the described method and system of the invention will be apparent tothose skilled in the art without departing from the scope and spirit ofthe invention. Although the invention has been described in connectionwith specific preferred embodiments, it should be understood that theinvention as claimed should not be unduly limited to such specificembodiments. Indeed, various modifications of the above-described modesfor carrying out the invention which are obvious to those skilled in thefield of molecular biology or related fields are intended to be withinthe scope of the following claims.

5 1 1872 DNA Homo sapiens 1 gaccctgaca cacccacctt ctcacctggg ctctgcgtatcccccagcct tgagggaaga 60 tgaagcctaa actgatgtac caggagaaag cccgctgggtcctgctggtc ctcattctgg 120 cggttgtggg cttcggagcc ctgatgactc agctgtttctatgggaatac ggcgacttgc 180 atctctttgg gcccaaccag cgcccagccc cctgctatgacccttgcgaa gcagtgctgg 240 tggaaagcat tcctgagggc ctggacttcc ccaatgcctccacggggaac ccttccacca 300 gccaggcctg gctgggcctg ctcgccggtg cgcacagcagcctggacatc gcctccttct 360 actggaccct caccaacaat gacacccaca cgcaggagccctctgcccag cagggtgagg 420 aggtcctccg gcagctgcag accctggcac caaagggcgtgaacgtccgc atcgctgtga 480 gcaagcccag cgggccccag ccacaggcgg acctgcaggctctgctgcag agcggtgccc 540 aggtccgcat ggtggacatg cagaagctga cccatggcgtcctgcatacc aagttctggg 600 tggtggacca gacccacttc tacctgggca gtgccaacatggactggcgt tcactgaccc 660 aggtcaagga gctgggcgtg gtcatgtaca actgcagctgcctggctcga gacctgacca 720 agatctttga ggcctactgg ttcctgggcc aggcaggcagctccatccca tcaacttggc 780 cccggttcta tgacacccgc tacaaccaag agacaccaatggagatctgc ctcaatggaa 840 cccctgctct ggcctacctg gcgagtgcgc ccccacccctgtgtccaagt ggccgcactc 900 cagacctgaa ggctctactc aacgtggtgg acaatgcccggagtttcatc tacgtcgctg 960 tcatgaacta cctgcccact ctggagttct cccaccctcacaggttctgg cctgccattg 1020 acgatgggct gcggcgggcc acctacgagc gtggcgtcaaggtgcgcctg ctcatcagct 1080 gctggggaca ctcggagcca tccatgcggg ccttcctgctctctctggct gccctgcgtg 1140 acaaccatac ccactctgac atccaggtga aactctttgtggtccccgcg gatgaggccc 1200 aggctcgaat cccatatgcc cgtgtcaacc acaacaagtacatggtgact gaacgcgcca 1260 cctacatcgg aacctccaac tggtctggca actacttcacggagacggcg ggcacctcgc 1320 tgctggtgac gcagaatggg aggggcggcc tgcggagccagctggaggcc attttcctga 1380 gggactggga ctccccttac agccatgacc ttgacacctcagctgacagc gtgggcaacg 1440 cctgccgcct gctctgaggc ccgatccagt gggcaggccaaggcctgctg ggcccccgcg 1500 gacccaggtg ctctgggtca cggtccctgt ccccgcacccccgcttctgt ctgccccatt 1560 gtggctcctc aggctctctc ccctgctctc ccacctctacctccaccccc accgggcctg 1620 acgctgtggc cccgggaccc agcagagctg ggggagggatcagcccccaa agaaatgggg 1680 gtgcatgctg ggcctggccc cctggcccac ccccactttccagggcaaaa agggcccagg 1740 gttataataa gtaaataact tgtctgtaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 1800 aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaaaa 1860 aaaaaaaaaa aa 1872 2 465 PRT Homo sapiens 2Met Lys Pro Lys Leu Met Tyr Gln Glu Lys Ala Arg Trp Val Leu Leu 1 5 1015 Val Leu Ile Leu Ala Val Val Gly Phe Gly Ala Leu Met Thr Gln Leu 20 2530 Phe Leu Trp Glu Tyr Gly Asp Leu His Leu Phe Gly Pro Asn Gln Arg 35 4045 Pro Ala Pro Cys Tyr Asp Pro Cys Glu Ala Val Leu Val Glu Ser Ile 50 5560 Pro Glu Gly Leu Asp Phe Pro Asn Ala Ser Thr Gly Asn Pro Ser Thr 65 7075 80 Ser Gln Ala Trp Leu Gly Leu Leu Ala Gly Ala His Ser Ser Leu Asp 8590 95 Ile Ala Ser Phe Tyr Trp Thr Leu Thr Asn Asn Asp Thr His Thr Gln100 105 110 Glu Pro Ser Ala Gln Gln Gly Glu Glu Val Leu Arg Gln Leu GlnThr 115 120 125 Leu Ala Pro Lys Gly Val Asn Val Arg Ile Ala Val Ser LysPro Ser 130 135 140 Gly Pro Gln Pro Gln Ala Asp Leu Gln Ala Leu Leu GlnSer Gly Ala 145 150 155 160 Gln Val Arg Met Val Asp Met Gln Lys Leu ThrHis Gly Val Leu His 165 170 175 Thr Lys Phe Trp Val Val Asp Gln Thr HisPhe Tyr Leu Gly Ser Ala 180 185 190 Asn Met Asp Trp Arg Ser Leu Thr GlnVal Lys Glu Leu Gly Val Val 195 200 205 Met Tyr Asn Cys Ser Cys Leu AlaArg Asp Leu Thr Lys Ile Phe Glu 210 215 220 Ala Tyr Trp Phe Leu Gly GlnAla Gly Ser Ser Ile Pro Ser Thr Trp 225 230 235 240 Pro Arg Phe Tyr AspThr Arg Tyr Asn Gln Glu Thr Pro Met Glu Ile 245 250 255 Cys Leu Asn GlyThr Pro Ala Leu Ala Tyr Leu Ala Ser Ala Pro Pro 260 265 270 Pro Leu CysPro Ser Gly Arg Thr Pro Asp Leu Lys Ala Leu Leu Asn 275 280 285 Val ValAsp Asn Ala Arg Ser Phe Ile Tyr Val Ala Val Met Asn Tyr 290 295 300 LeuPro Thr Leu Glu Phe Ser His Pro His Arg Phe Trp Pro Ala Ile 305 310 315320 Asp Asp Gly Leu Arg Arg Ala Thr Tyr Glu Arg Gly Val Lys Val Arg 325330 335 Leu Leu Ile Ser Cys Trp Gly His Ser Glu Pro Ser Met Arg Ala Phe340 345 350 Leu Leu Ser Leu Ala Ala Leu Arg Asp Asn His Thr His Ser AspIle 355 360 365 Gln Val Lys Leu Phe Val Val Pro Ala Asp Glu Ala Gln AlaArg Ile 370 375 380 Pro Tyr Ala Arg Val Asn His Asn Lys Tyr Met Val ThrGlu Arg Ala 385 390 395 400 Thr Tyr Ile Gly Thr Ser Asn Trp Ser Gly AsnTyr Phe Thr Glu Thr 405 410 415 Ala Gly Thr Ser Leu Leu Val Thr Gln AsnGly Arg Gly Gly Leu Arg 420 425 430 Ser Gln Leu Glu Ala Ile Phe Leu ArgAsp Trp Asp Ser Pro Tyr Ser 435 440 445 His Asp Leu Asp Thr Ser Ala AspSer Val Gly Asn Ala Cys Arg Leu 450 455 460 Leu 465 3 16063 DNA Homosapiens 3 ggaggatcac ttgagtccag gagttcagcc tgggcaataa gcaagaccccgtctatacaa 60 aattaaaaaa aaaaaaatta gctgcggctg ggcgcggtgg ctcatgcctgtaatcccagc 120 acttttgggg gcctagacgg gcggatcacg aggtcaggag atcgagaccatcctggctaa 180 cacggtgaaa ccccatctct actaaaaaat acaaaaaaaa aattagccgggcatggtggc 240 gggcgcctgt agtcccagct actcgggaag ctgaggtggg cgaattgctcgaaccaggga 300 ggtggaggtt gcagtgagcc gagatggcgc cactgcactc cagcgtgggagagacagagc 360 gagactgcgt ctccaaaaaa aaaaaaaaaa gcagctgggc atggtggcacgtgcctgtag 420 tcccagctgc ttgggaggct gaggcaggag gattgcttga gcctgggaagttgaggccgc 480 agtgagctat gatcatgctg ctgcactcca gcctagggaa cagaactctggcactaaaaa 540 aagaaaagaa aagaaaagaa aataggctta caataaaaag caatcttgacctaccccaaa 600 atcccactcc cttgaagaca accacttctg ccctgttgct tctctcagtaatactccttt 660 tctgcagtta tctcttgact tcctgccatg tgtgtttggg ggagcttgagggaagagggt 720 gtccctgtct cattgagaga ggtgccaccc tgccgtgaaa agaggtgccaccctctgtca 780 attaggagag gagggatgag agaggaaagg ggtctccagt gtatttctccagccggggcc 840 ttaaatccct cttgggagat atgggatggg gtggatcgga aaataaatttttttaaatcc 900 ctaccaaaat atcagctggc tttttttaaa aaatcaaata ccaaaatctaaatagactcc 960 aacagaaaat tcaccatctc ctctgacctt ttcttcccat ctcatgctgtgaactgtctt 1020 ctgttgactt tatcgctacc tttcttcatt ctgttattca accatgatctctccgtttca 1080 ttttataagc gttttattaa tttcatttat gtatttattt ttgactaggtaatgcatgtc 1140 catggtacac aaattcacaa ggtttgtaaa tgagaaaaga cgtgaggttccttttgttct 1200 ttacctgtgg cctccctgcc ctacacgggg actctagggt ggaatgtagcaaagcccatc 1260 caccagccat gtactacccc ccaacccggc caggctggag cgaccgtgtctggggagccg 1320 agccccgctt ctcgctgcgg tgagcccgga ctggggcacg cactgcgcagactccccgct 1380 gcagtgggcg gagctcccac aggccccgcc ccctcctccc accctcgttcagcctgtcca 1440 gacagaagct ggggcccagc ggaggtagca gcagacgcct gagagcgaggccgaggcccc 1500 tcaggggtag gtggggggag gctggctggg gggatgggca gcggggtggcagggaggccc 1560 cgatgatccc tggctgagct gtgtggtgtg ggggcacctg agggcggaggagccacctag 1620 aatatacccc ctgtcccagg gctgagaaaa gccaggcaca aagacctggggctccgctga 1680 cctcatccag cgctgccttt ctgggggtgt ctacaggttg gaggagggagaagggggctg 1740 ctagagggga gctgcgtggg tgctgatggg ggcagggtgg gcactgcagagtgaagagca 1800 ggcagcagag cgttggattt tgagggattt cgaaatcctg ctgaccagtgaccagcttcc 1860 tctttttggg gtggtgggca gtgtgccagt ggcccccatg attatgaatagccccaagac 1920 taatcactct tctgtgatgt ctgtccctgg aaagcgtaga acaccccacacagtgcccac 1980 ttttgttctg ccagtttgga gaccctgaca cacccacctt ctcacctgggctctgcgtat 2040 cccccagcct tgagggaaga tgaagcctaa actgatgtac caggaggtaggtggattggg 2100 gggctcagca gggtggaact gggtacagtg ggggtggggg ttcccaagagccacctgtca 2160 cccccgttgt tgtccccatc cccagctgaa ggtgcctgca gaggagcccgccaatgagct 2220 gcccatgaat gagattgagg cgtggaaggc tgcggaaaag gtaggagccctccgccaccc 2280 tcgctctgtc tcagagacag gctgggctgc ccccctcggg ctggctgaccacctcctcct 2340 ccccacagaa agcccgctgg gtcctgctgg tcctcattct ggcggttgtgggcttcggag 2400 ccctgatgac tcagctgttt ctatgggaat acggcgactt gcatctctttgggcccaacc 2460 agcgcccagc cccctgctat gacccttgcg agtaagtggg gggtgctgcacttggtgggg 2520 gaggggcctg ccagaccagg tacacttaag cacacactaa acagggcctgcactcagccc 2580 tacccagcgc ttgcgacaag tgaggaggtt gcaggctccc aagtgctcgcccgccccctc 2640 ctcctccaca cacatagttt ctatggcagc cacagcgtca tcttctgtcaggcctgtgaa 2700 cagacacagc atcttccacc cacatctgtg gacccacaca cacatctcaatacacgacct 2760 tccttccaca cctctagaca gacacgcaga ggatcatgag tccaggcacgcattcaaata 2820 cacacagttt taaaaaattt tttttaaaag aaaagaaaaa ctcaaatacagtttagctgg 2880 gcttggtggc tcacgcctgt aattgcaaca ctttgcgggg ctgaggcgggaggattgctt 2940 gagcccagga gttccagacc agcctgggca atgtagtgcg gctccatctctactaaaagt 3000 aaaaaaagta accaggcata ctggtgcaca cctgtagtcc cagctactcaagaggttgag 3060 gcgggaggat cgcttgagcc caggggttcg aggctgcagt gagctgagatggcgccactg 3120 cgcttccagc ctgggggaca gggcaagact ctgtctctaa aaaaaaaaaaatacagtcta 3180 tccaacacac ccatggacgg acagctgagc actcacctcc cagcccttgctctccggcac 3240 cgtatggctg atagcatccc ccacccccca gagcagtgct ggtggaaagcattcctgagg 3300 gcctggactt ccccaatgcc tccacgggga acccttccac cagccaggcctggctgggcc 3360 tgctcgccgg tgcgcacagc agcctggaca tcgcctcctt ctactggaccctcaccaaca 3420 atgacaccca cacgcaggag ccctctgccc agcaggtacc tgcaaccttggccctggccg 3480 gcagcagggg cagggggtgg gaggcagcgg gggctgtggg gaatgaaggggtttctcctg 3540 cagcccagga gacagagggg tgtgtctcac acagcagatt ggacacaggtgtttgcaagc 3600 agctgtcgtc acgtggctct ctggactggg ggcgtttgtc acggtcatctgtaggcctct 3660 gaatgtcagg gtgcgggttt ggttacaagg gattattagg ctggcagatgtcactcaccc 3720 acaagctgtc ctgacccagt cacacaaaga aagggaagag tggagtaattaacagcccag 3780 cctcaagaca ggggccacct cccagctgtg tgaccccagg caggccacctcttctctcca 3840 gcttcagttt ctttatctgt aaaatggggc caatttatag cacctgcttcttagggctct 3900 tgtgaggatg aaatggacta atccatgcaa attttagcac agtgcctggcacagagtcag 3960 cctttgtgag tctgctgtta ctatatatcc tggtatggtc tgcagacaaacttaaagaac 4020 ataaaagctt cacaatttga aaaggaacag cctacatgga taacctcttccattgaaaaa 4080 ccatgaattt gttccttctg ttttcttctc cctactggct ctttttgtgagaagttgtct 4140 taaaacttaa ctaaaattac aaggctcctt aagaactgcc tgaaaaaaataattatggcc 4200 aagtgtggtg attcacacct gtaatcccag cactttggga ggcctagacaggaggatcac 4260 tggagcccaa gagttcgaga ctagcctgag caacatagtg agacccgccccccacctccc 4320 ctcaaccatc tctactaaaa ataaaaaaaa attaggaggg tgttgtggtgcatgcctgca 4380 gtctcagata ctcaggagat ggaaggagga ggatggcttg agcccaggagttggaggctg 4440 cagcgagccg tgatcatgcc actacactcc agcccaggca acatagcaagaccctgtctc 4500 aaaaaaaaga aaaagaaaaa gtaataataa taattacaaa agttaaaaaccaaagccagg 4560 catagtggta cacacctgtg gtcccagcta cttgggaggc tgaagcaggagaatgacttg 4620 agcctaggaa ttggaggctg cagtgagctg tgatcatgcc actgcactctagcctgggca 4680 acatagtgaa accctgtctc taaaaaaaaa tttttttatg ttaaaaaaccgtatgagctc 4740 ccactccctg ctggctccct ctaaagtggt ttaaaacaca gatgtaggaggcagatggtc 4800 tgggttcaaa tcctgctcca tagctggatg tggtggtgca ctcctgaggtactagctact 4860 tgggagactg aggcaggcgg aatgcttgag cccagggatt gaagatcagcctgggcaaca 4920 tagcaagtcc ctgtctcaac aaacaaacaa acaaaaaaac aaaaacaaaatcccactcca 4980 ctactgagtt ctctgtgggg attataatta aaccagcctc actgggttgtgagaattcag 5040 tgagttcgct gagaagaggc ctagaacagg gcctggcaca cagtaggttccagggcatcc 5100 ttgactgttg ctgttgttgg catcatcgtg cctcacccga taccttccaggaccccctgc 5160 ctgagcctcg cccccaccat actgggagat gcctggaggc cctgccttgatgctgaattt 5220 tgagaaagtc cctggagggg caggaggggt caggaggact tggagggggatcacagggca 5280 actaattatt aaagcagata aagatgttta aaacagataa ggaagtcttttaatatttta 5340 atctgtaaag tctttaatct atgcaggcta atgtaaagtc tgtttactcctaatcatgtc 5400 tcaaaataac tccaccgggc attaccttgt ggggttggag agctggctggtccagcccct 5460 cagaagctct cccctccccg cagggtgagg aggtcctccg gcagctgcagaccctggcac 5520 caaagggcgt gaacgtccgc atcgctgtga gcaagcccag cgggccccagccacaggcgg 5580 acctgcaggc tctgctgcag agcggtgagc tggggcccaa ctggggctggtctgggcctg 5640 ggggtaccca gcctggcccc tgatctctgc ccctgctggt cacaggtgcccaggtccgca 5700 tggtggacat gcagaagctg acccatggcg tcctgcatac caagttctgggtggtggacc 5760 agacccactt ctacctgggc agtgccaaca tggactggcg ttcactgacccaggtctgtc 5820 tgcaccctgt ctaccttcct tccaggccac tccctgcccc acagggcacccagcctccga 5880 ctgcatccct cactcaatcc agagtcctct ccacccattc tctgtaatggcttccttctt 5940 gcctcctacc aggcctccct aatccaagcc atgcacggtg gctcacacctataatctcaa 6000 cactttggga ggccaaggtg ggaggattgc ttgagcccag gagttggagaccagcctggg 6060 caacatagtg agaccccatc tctaccaaaa aaaaaaaaat aagcccggtgtggtggcaca 6120 cacctctggt cccagctcct tgggagactg aggtgggagg atcacttgagcccaggagtt 6180 tgaggctaca gtgggttgta ttcatgccac tgcactccag cctgagtgacagagtgagac 6240 cctgttacaa aacaaagaat ccctaaatgg agccctctac tgccctccccctgctcctgg 6300 aagcctgggg ctccctctga tccccaattg cagctaccag cccctctacatggcattcaa 6360 gaacctgcgc acccatttga tcttcattgt acatctctgt gtgcctgctctaagtccagc 6420 cctatcctgg gtaatgttgg gaacatggtg gtaacagatg gacctcatggaactcccagc 6480 ccaatgcaga ctggcctgtc acctgacagt gacagcccag aggggtcagggccggggttg 6540 gggagacaca ggcagagggt cagggccagg atggagggaa cagagggctgtggaagctca 6600 gagaccccaa cctggggcat tggagggttt cccaaaggag gtataactaatctgatccct 6660 gaaggatagg gaggaattag cgcaagatgg aacaggaaac agcttgggcaatgaggtgaa 6720 gataagacag gacaataact catgaattca tttcctcaac agatagttcccctaaccttt 6780 aatctcagca attatgcagg gagatgctgt agatatagct gtgactgagacatccctagt 6840 gcctgtcctc ccagcccaat gggaagacca gtttgtcacc agaaagaatgaggagggaat 6900 cccaagggac tgtgagagcc cagaggaatg cctggtgcag gctgggtagtcacgggaggc 6960 ttcctgaagg aggcaacatg tcagcctaga cctaagaatg agtagaagctagctcagtgg 7020 agggtagaag caacagcaga ttgcaaacgt tcaggaaacc tggagctttggaataactga 7080 ttttcatcaa aacttaagtt gataatcatt ctaggacttt agctattggagctggggtgg 7140 agggggtact gtgggacagg gggaaagaca gagaccagac tggggagtgtccctgtcatc 7200 tgtgagcact aggccgctat cgctgagctc agcactgccc tcctacaggtcaaggagctg 7260 ggcgtggtca tgtacaactg cagctgcctg gctcgagacc tgaccaagatctttgaggcc 7320 tactggttcc tgggccaggc aggcagctcc atcccatcaa cttggccccggttctatgac 7380 acccgctaca accaagagac accaatggag atctgcctca atggaacccctgctctggcc 7440 tacctggcgg tgagtctggg gcaagtgggg cctgtcatgt cccagccccatgccgtcact 7500 cacagcctcc atctgtccct gtttggtgat gacagggagg gcgtatcctgaccatcagtt 7560 ctcaccccag ctcattctgc ttggtcaggg gcctggagta gttcccaacatccctcggcc 7620 tctatttcag ttagaaaatg ggtattgttt ccaacctgtt agggctgctgggagaggtac 7680 cctgggttca tgcacaccaa acctttggtg ctctatatca tccagtatagccacaggtgg 7740 ctctttcagt ttaagttaat taaatgcaat taacaattca ggccaagtggggtggcttat 7800 gcctgtaatc ccaatacttt gggaggttga ggtgacagga tcacttgaggccaggagttt 7860 gagaccagcc tggacaacat agcaagactc catctttaca aacaaacaaacaaacaaaca 7920 aaactagctg ggttgggttg tgcatgcatg tagtcacagc tacgcaggaggctgaggcag 7980 ggggatcact tgagcccagg aaatctaggc tgcagtgagc catgatcacaccactgtact 8040 ccagcctggg tgacagcctg tttcaaaaaa aaattgtttc aagccaggcatggtttctca 8100 tccctgtaat cccagcactt tgggaggcca aggccaagat gggaggatcacttgaggcca 8160 ggagttcatg accagcctgg gcaacatagg gagacatcat ttctttctattttttttttt 8220 ttttggtctt attatttatt gttctagata ggatacccaa gaactagggagacaccattt 8280 ctacaaaaac ataatattaa ttaaaattag ccgggtgtgg ttgtgtgcacctgtagtccc 8340 tggggaggct aagacagggg gatcacttga gcccaggagt ttgaggctgcgtgagctgat 8400 tgtaccactg cactccagcc tgggcaagag gctgttgccc tgtctccaaaaaaagaaaaa 8460 attcagttcc tcatgcagta gccacatttc atatgctcag tagcacctgtagcaagtgac 8520 caccatattg gacatttcca tcactgtgac aggctctgtt ggacaacactggctctcgcc 8580 atggcagata ctgatcactc tggacaaggc actgatgttt ctagctcttgatagtttcac 8640 tagttgaggc aggcaaccca ggtctcccta ggtccccctg agcaagttacctgtccaagc 8700 ccagagtcat cgtggaaggc acaaccctaa ggcgtggacg tagggaagtgtgactcattg 8760 gggtctttca ctacaagggc ctcccgcagg ggatcaaggc tctcctcattaccacttccc 8820 cttttagagc ctcagtttcc ttgtctcttg agcattaagg aagatggggggccaggcaca 8880 gtggctcatg cctgtaatcc tagcattttg ggagtccagg atgggcggatcacttgagct 8940 caggggttgg agaccagcct gggaaacgtg atgaaacccc atctctaccaaaaatacaaa 9000 aattagcctg gaggggtggc gggcacctgt aatcccagct actcgggaggctgaggcagg 9060 agaatcactt gaacccagga ggtagaggtt gcagggagcc gagattgcaccattgccctc 9120 caggctgggt gacagagtga gactccactt caaaaaaaaa aaaagggggggaagcggggg 9180 agcgggggaa ctgggaagag ggcctggtga ggcactgggc acccgaggggttcccagtca 9240 aggcaggctg tgagcaaatc agggaagaaa gtgactcgag gctgggcacagtggctcacg 9300 cctgtaatcc cagcactttg ggaggcctag gcaggtggat tgcctgaggtcaggagttcg 9360 agacctgcct ggccaacatg gtgaaatccc atctccacta aaaatacaaaaaaattagct 9420 ggctatggtg atgtacgcct gtagtcccag ctacttggga ggctgaggcaggagaatcgc 9480 ttgaacccaa gaggcagcag aggttgcaat gagctgagat catgccactgcactccagcc 9540 taggagacag agcaagactc catctcaaaa aaaaaaaaaa aaaaaaagacttgagcagag 9600 gtcctcaaac tgagcatgcc ccagaatcag ctgggcgggc ttgttaaaacccagattcct 9660 ggaccccacc ccagcactct gattcagtag gccatgtgca gtgaggcccaggaatttgca 9720 tttctaacaa gttcccaggt gatgctgttg ccgctggatc agggaccttacgttgagaag 9780 cactgggtta gagcgtaaat tctggaacca gacagcctgg gttcgaatcctggctccatt 9840 tatctgctgt gtgactttaa gtaagtcact taccctctct gagcctcagtttcctcacat 9900 gtgaaatgga tgtggtgatt gaccctcttc ttcatggagg ctgaggatttggtgagatcc 9960 acagtacctg gcttgtggtg agctgtccgt atgtggggtc cgttgtgacgatgaccctgg 10020 cagggcacat gtcttaactg tcccctcgcc ctcagagtgc gcccccacccctgtgtccaa 10080 gtggccgcac tccagacctg aaggctctac tcaacgtggt ggacaatgcccggagtttca 10140 tctacgtcgc tgtcatgaac tacctgccca ctctggagtt ctcccaccctcacaggtact 10200 gctgggtgtg gagataggga gccgctgcag ttggccagga gacgggagagggaatcatgg 10260 agaccagaaa gctggtgggg gctccaggca aggggacaga tggaagagaagctgcaggga 10320 gagacagtca ccaggaggtg accggaagaa ggtatctagg cacttgagacaggagaaaga 10380 gagattacag aggagacagg gatgaggttt caggacaagg tttgagggaacagagaaaag 10440 gatgagaggg ccgggcgtgg tggctcacgc ctgtaatccc aacattttgcggggctgagg 10500 tgggtggatc acttgaggtc aggagttcaa gaccagcctg gctaacatggtgaaatccca 10560 tctctcctaa aaatacaaaa attagccggg cgtggtggca cgtgcttgtaatcccagcta 10620 cttgagaggc tgaggcagga gaattgcttg aacctgggag gtgaaggttgcagtgagttg 10680 agatcgcgcc actgctctcc agcctgtgcg acagacagag caagactctgtctcaaaaaa 10740 acaacaaaaa aaaagagaag gctcagaata ttggggttga gggcaggaagcctgaggcag 10800 gggtgcagga tgtgggattt ggggaggtag gaggcatggg ctggaaacaggatgaggggc 10860 ttgggggatg gggactaaaa gtatttgggt ttagggtagc aagcttggggatttgtgatc 10920 ctgggataag aaggataaca accggccgga cgtggtggct cacacctgtaatcccagcac 10980 tttgggaggc tgaggcgggt ggatcacgag gtcaggagat cgagaccatcctggccaaca 11040 tggtgaagcc ccgtctctac taaaaataca aaaaattagc caggtgtggtggcaggcgcc 11100 tgtagtccca gctacttggg aggctgaggc aggagaatcg cttgaacccgggagtcggag 11160 gttgcagtga gccaagatca tgccactgca ctccagcctg ggcgacggagcgagacacct 11220 tctcaaaaaa aaaaaaaaga aaagaaaaaa aaaaagaagg ataacaaccatacccactgc 11280 aacatccagg tggatgatgg cacttgtggg gctcaaagaa ggtattctaggggcagtaga 11340 taagacagtg ggtccaggca tggtggctca cgcctgtaat cccaacactttgggaggccg 11400 aggcggaagg atccctagga gtttgagacc agcctgggca acataatgagaccccgtctc 11460 tatagaaaaa ttggaagatt agcccagtgc ggtggcactc acctgcagtcccagctactc 11520 aggagactga ggcaggagga tcacttgagc ccaggagttg gaggctgcattgagctatgg 11580 tcgtgccact acactccagc ctgggtgata gagcaagaac ctgtctcaaaaagaaaaaaa 11640 agaggatgga ccgggcacag tggctcacgc ctgtaatccc agcactttgggaggccaagg 11700 cgggcagatc acctgaggtc gggagttcga gaccagcctg acaaacatggagaaaccccg 11760 tctctactaa aaatacaaaa ttagccaggc gtggtagcgc atgcctgtaatcctagctac 11820 ccgggaggct gaggcaggag aatcgcttga acccaggagg cagaggttgcagtgagccga 11880 gattgtgcca ttgcactcca gcgtgggcaa caagagcgaa attccacctcaaaaaaaaaa 11940 agaaagaaag aataaaagag gatgacaaca ctgggttttg gggagcaggaatgggagcca 12000 cagccaggaa gaggaaatag ggatgtggga tttatggaga caggaacagggtctgggagc 12060 cagcgatgag gaagtcctct caatagctaa agcagggccc aggcttggttccccaaagct 12120 gagggcaaag cctgtggaca cagccgccct ctgcatcctg ccccacctcctatacacccg 12180 tcctcaggtt ctggcctgcc attgacgatg ggctgcggcg ggccacctacgagcgtggcg 12240 tcaaggtgcg cctgctcatc agctgctggg gacactcgga gccatccatgcgggccttcc 12300 tgctctctct ggctgccctg cgtgacaacc atacccactc tgacatccaggtggtaagta 12360 ctgccccaag ccaccccttg gcccctgtgt ggggcagtcc tagggacacagccctcatgg 12420 gactcgtctg tcaatgacaa gggcagccca gagtgagccc tgtcactgtggggaaaccat 12480 gggtcagggc cagggtcatg gggcacaggg agaggggcca ggaccgggatgagggggcac 12540 aggcagaggg gttgggaccg ggatgggggt gcagagagag gtgttgggaccaggaatggg 12600 gacatgggca ggtagagggg tcagggctgg gattgggagc acaggcagaggggtcagggc 12660 tgggatgggg aggcacaggc agaagggtcg gggccgggtt ggagggcacagggagagggg 12720 ttagggccag ggttgggggc acaggcagag gggtcggggc caggatggaggaggcccagg 12780 cagaggggtg agggcttggg gtggggggca cagagagagg gtttgggaccaggactgggg 12840 gagtgggcag gtagaggggt cggggctggg gttgggggca caggcagaggggtcagggct 12900 gggatggagg aggtacaggc agaggggtca gggctgggat gggggggcacaggcagaggg 12960 gtcagggctg ggatggagga ggcccaggca gaggggtcag ggcccagggtcggggggcac 13020 aggcagaggg gtcagggctg gggtggggat gcccagagga agcctctgccctagcgggaa 13080 gggccaagga agatgttctg gaaatggggg catctgagat gagacctcaggaatgaacag 13140 gagccattct gccgggaaca gtgttttgca aatgagacca ccggggcctccctttcagct 13200 ttcgttctca gagggcccct ccacctggcc ctgttctggc ccccgaggattctgtgggaa 13260 gcagtggagt cccacagatc tcgctccaca ctctgctccc tgatcccggggctcctccga 13320 ctccccctgc ctctcacact ccttcccatc ctcccctccc actcagaaactctttgtggt 13380 ccccgcggat gaggcccagg ctcgaatccc atatgcccgt gtcaaccacaacaagtacat 13440 ggtgactgaa cgcgccacct acatcggtga gtgtcttgag caccacggggcgctgaagaa 13500 gagggggttc agacaccagg ggcggccccc cgagggtgcc cttatgctccacccattcct 13560 ctctaggaac ctccaactgg tctggcaact acttcacgga gacggcgggcacctcgctgc 13620 tggtgacgca gaatgggagg ggcggcctgc ggagccagct ggaggccattttcctgaggg 13680 actgggactc cccttacagc catgaccttg acacctcagc tgacagcgtgggcaacgcct 13740 gccgcctgct ctgaggcccg atccagtggg caggccaagg cctgctgggcccccgcggac 13800 ccaggtgctc tgggtcacgg tccctgtccc cgcgcccccg cttctgtctgccccattgtg 13860 gctcctcagg ctctctcccc tgctctccca cctctacctc cacccccaccggcctgacgc 13920 tgtggccccg ggacccagca gagctggggg agggatcagc ccccaaagaaatgggggtgc 13980 atgctgggcc tggccccctg gcccaccccc actttccagg gcaaaaagggcccagggtta 14040 taataagtaa ataacttgtc tgtacagcct gtgcctgact gagtggtgtgagatggggtg 14100 caggggtagg ggacagctgg catgggcctc tggtggggac atctttttgtgctgagccct 14160 caacatgtca ctggcatgtg ctgagccctc agtgtgtgac cagtgtgggttagcatgtac 14220 tgagccctca gcatgtgctg gcatgggttg gcatgtgctg agcccttaacatgtagtgta 14280 catgggctga cctgtgctga gcatgcagcg tgccaccctg tggcccggcatggacttagc 14340 actcatgtag ccagcatggg tatgtgctgc agagaagcat gttcccagattgatcagcag 14400 ggaccaaacc attgccacat cccaagggtg aacaagcatg gctgagcaccagagtgtgca 14460 ccaagtgtga atttaggcct gccaagtgga tttacaccca gcacgtcccaaatgtgggtg 14520 agtgcatgcc aaccctgtaa acatgtagga aggacaggtc aacagacaaggagaccccag 14580 catccggcaa acttgattaa cacatactga acacagcatg ttctgagggtggattcccaa 14640 cacgccaagc acatagcgta tttaggacaa gggttagtca accaagcacagcttttccct 14700 cctagtgtga cagcagccca ggctcccgcc taagccagtg gtcagctgggcccagcatcg 14760 cagagcaagt cactgggtgc cagcctggag cccccattcc ccccagggccagtccaagcc 14820 ccaggcagtg agagcaggct tgaagcagga ctgctgaaca gttctatattgaaatagaca 14880 gaggcagcag ggccaagggc gagcgcaggg ccagcggggt gcagccccctttcctgctgt 14940 ccttctggcc aaggagcatg ggccagactc caaagccctg ctgtgtttaggagaggtgtg 15000 caggcacgca ccgcaccgca gacggggaat gagaatttct ggataactatctttctgtaa 15060 gaataatttg tgggttcagg agatggctct gaggagcagt tcaggttgggagggaatgcc 15120 agcccagcta gcgcagcccc cagtgatggg caggggtgga atcaccatcagtggtgcccg 15180 gtgacatgct ggaggaagct ggtggccccc cggggtggac catgctggtgggagcggcgg 15240 ggtgggagcc cctgagcccg tgggcccctg acgctctcca gggtgcagtctggctcactc 15300 agccattctc caggacagct gctggggtcg aagagctcag ggtctggcctctgtggggga 15360 agagagaggg gcatcagcca agcagtgtta gtgtattagt gtctgctgagcctctgtcac 15420 ctcctcctga tgagggtgag catgtgctag ggtcttacca gtgctgggcctttattgccc 15480 ctttgtagtg atgtgatgac agctcactgc agcctcgacc tccctggctcaagtgatcct 15540 cccacctcag cctcctgagt agctgggact acaggtgcat gccaccaaaaccagctaata 15600 tttctaattt tttggtgcag acgggatctc actatgttgc ccaggctctttatcaagctt 15660 cttataagta gattactgtg ccctgagttt ttgtcacagc ctggaagtagctgagtaagt 15720 gctgagcctt atcatatctt gacggtatta ggtgtgtgct gagcctcctaagtccctaat 15780 tgtatcctga acatgggtga gtgtgtgttg agcctcctca cacactaatcatatcctgag 15840 cacgggtgag tgtgtgctga gcctcctccc accctaatcg tatcctgaacgcacccggct 15900 cacctcagct tccatggtca tgttgtcaat gttgggccca tcctcatctcgctgcaggat 15960 ggccagtggc tcagcagagg ccccgagatg ctctccttcc tcccagctgctgccccggca 16020 gggcctgtca tcctcaggcg agacctggct cagccgaatg agg 16063 4437 PRT Homo sapiens 4 Met Thr Gln Leu Phe Leu Trp Glu Tyr Gly Asp LeuHis Leu Phe Gly 1 5 10 15 Pro Asn Gln Arg Pro Ala Pro Cys Tyr Asp ProCys Glu Ala Val Leu 20 25 30 Val Glu Ser Ile Pro Glu Gly Leu Asp Phe ProAsn Ala Ser Thr Gly 35 40 45 Asn Pro Ser Thr Ser Gln Ala Trp Leu Gly LeuLeu Ala Gly Ala His 50 55 60 Ser Ser Leu Asp Ile Ala Ser Phe Tyr Trp ThrLeu Thr Asn Asn Asp 65 70 75 80 Thr His Thr Gln Glu Pro Ser Ala Gln GlnGly Glu Glu Val Leu Arg 85 90 95 Gln Leu Gln Thr Leu Ala Pro Lys Gly ValAsn Val Arg Ile Ala Val 100 105 110 Ser Lys Pro Ser Gly Pro Gln Pro GlnAla Asp Leu Gln Ala Leu Leu 115 120 125 Gln Ser Gly Ala Gln Val Arg MetVal Asp Met Gln Lys Leu Thr His 130 135 140 Gly Val Leu His Thr Lys PheTrp Val Val Asp Gln Thr His Phe Tyr 145 150 155 160 Leu Gly Ser Ala AsnMet Asp Trp Arg Ser Leu Thr Gln Val Lys Glu 165 170 175 Leu Gly Val ValMet Tyr Asn Cys Ser Cys Leu Ala Arg Asp Leu Thr 180 185 190 Lys Ile PheGlu Ala Tyr Trp Phe Leu Gly Gln Ala Gly Ser Ser Ile 195 200 205 Pro SerThr Trp Pro Arg Phe Tyr Asp Thr Arg Tyr Asn Gln Glu Thr 210 215 220 ProMet Glu Ile Cys Leu Asn Gly Thr Pro Ala Leu Ala Tyr Leu Ala 225 230 235240 Ser Ala Pro Pro Pro Leu Cys Pro Ser Gly Arg Thr Pro Asp Leu Lys 245250 255 Ala Leu Leu Asn Val Val Asp Asn Ala Arg Ser Phe Ile Tyr Val Ala260 265 270 Val Met Asn Tyr Leu Pro Thr Leu Glu Phe Ser His Pro His ArgPhe 275 280 285 Trp Pro Ala Ile Asp Asp Gly Leu Arg Arg Ala Thr Tyr GluArg Gly 290 295 300 Val Lys Val Arg Leu Leu Ile Ser Cys Trp Gly His SerGlu Pro Ser 305 310 315 320 Met Arg Ala Phe Leu Leu Ser Leu Ala Ala LeuArg Asp Asn His Thr 325 330 335 His Ser Asp Ile Gln Val Lys Leu Phe ValVal Pro Ala Asp Glu Ala 340 345 350 Gln Ala Arg Ile Pro Tyr Ala Arg ValAsn His Asn Lys Tyr Met Val 355 360 365 Thr Glu Arg Ala Thr Tyr Ile GlyThr Ser Asn Trp Ser Gly Asn Tyr 370 375 380 Phe Thr Glu Thr Ala Gly ThrSer Leu Leu Val Thr Gln Asn Gly Arg 385 390 395 400 Gly Gly Leu Arg SerGln Leu Glu Ala Ile Phe Leu Arg Asp Trp Asp 405 410 415 Ser Pro Tyr IleHis Asp Leu Asp Thr Ser Ala Asp Ser Val Gly Asn 420 425 430 Ala Cys ArgLeu Leu 435 5 488 PRT Mus musculus 5 Met Lys Pro Lys Leu Met Tyr Gln GluLeu Lys Val Pro Val Glu Glu 1 5 10 15 Pro Ala Gly Glu Leu Pro Leu AsnGlu Ile Glu Ala Trp Lys Ala Ala 20 25 30 Glu Lys Lys Ala Arg Trp Val LeuLeu Val Leu Ile Leu Ala Val Val 35 40 45 Gly Phe Gly Ala Leu Met Thr GlnLeu Phe Leu Trp Glu Tyr Gly Asp 50 55 60 Leu His Leu Phe Gly Pro Asn GlnArg Pro Ala Pro Cys Tyr Asp Pro 65 70 75 80 Cys Glu Ala Val Leu Val GluSer Ile Pro Glu Gly Leu Glu Phe Pro 85 90 95 Asn Ala Thr Thr Ser Asn ProSer Thr Ser Gln Ala Trp Leu Gly Leu 100 105 110 Leu Ala Gly Ala His SerSer Leu Asp Ile Ala Ser Phe Tyr Trp Thr 115 120 125 Leu Thr Asn Asn AspThr His Thr Gln Glu Pro Ser Ala Gln Gln Gly 130 135 140 Glu Glu Val LeuGln Gln Leu Gln Ala Leu Ala Pro Arg Gly Val Lys 145 150 155 160 Val ArgIle Ala Val Ser Lys Pro Asn Gly Pro Leu Ala Asp Leu Gln 165 170 175 SerLeu Leu Gln Ser Gly Ala Gln Val Arg Met Val Asp Met Gln Lys 180 185 190Leu Thr His Gly Val Leu His Thr Lys Phe Trp Val Val Asp Gln Thr 195 200205 His Phe Tyr Leu Gly Ser Ala Asn Met Asp Trp Arg Ser Leu Thr Gln 210215 220 Val Lys Glu Leu Gly Val Val Met Tyr Asn Cys Ser Cys Leu Ala Arg225 230 235 240 Asp Leu Thr Lys Ile Phe Glu Ala Tyr Trp Phe Leu Gly GlnAla Gly 245 250 255 Ser Ser Ile Pro Ser Thr Trp Pro Arg Ser Phe Asp ThrArg Tyr Asn 260 265 270 Gln Glu Thr Pro Met Glu Ile Cys Leu Asn Gly ThrPro Ala Leu Ala 275 280 285 Tyr Leu Ala Ser Ala Pro Pro Pro Leu Cys ProSer Gly Arg Thr Pro 290 295 300 Asp Leu Lys Ala Leu Leu Asn Val Val AspSer Ala Arg Ser Phe Ile 305 310 315 320 Tyr Ile Ala Val Met Asn Tyr LeuPro Thr Met Glu Phe Ser His Pro 325 330 335 Arg Arg Phe Trp Pro Ala IleAsp Asp Gly Leu Arg Arg Ala Ala Tyr 340 345 350 Glu Arg Gly Val Lys ValArg Leu Leu Ile Ser Cys Trp Gly His Ser 355 360 365 Asp Pro Ser Met ArgSer Phe Leu Leu Ser Leu Ala Ala Leu His Asp 370 375 380 Asn His Thr HisSer Asp Ile Gln Val Lys Leu Phe Val Val Pro Thr 385 390 395 400 Asp GluSer Gln Ala Arg Ile Pro Tyr Ala Arg Val Asn His Asn Lys 405 410 415 TyrMet Val Thr Glu Arg Ala Ser Tyr Ile Gly Thr Ser Asn Trp Ser 420 425 430Gly Ser Tyr Phe Thr Glu Thr Ala Gly Thr Ser Leu Leu Val Thr Gln 435 440445 Asn Gly His Gly Gly Leu Arg Ser Gln Leu Glu Ala Val Phe Leu Arg 450455 460 Asp Trp Glu Ser Pro Tyr Ser His Asp Leu Asp Thr Ser Ala Asn Ser465 470 475 480 Val Gly Asn Ala Cys Arg Leu Leu 485

That which is claimed is:
 1. An isolated polypeptide having an aminoacid sequence consisting of SEQ ID NO:2.
 2. An isolated polypeptidehaving an amino acid sequence consisting of SEQ ID NO:2.
 3. Acomposition comprising the polypeptide of claim 1 and a carrier.
 4. Acomposition comprising the polypeptide of claim 2 and a carrier.